SummaryElectrophoretic variants of mitochondrial aldehyde dehydrogenase (AHD‐A2) are widely distributed among inbred strains ofMus musculusand have been used to localize the gene encoding AHD‐A2(Ahd‐1)at the non‐centromeric end of chromosome 4.In the mouse(Mus musculus),aldehyde dehydrogenase (AHD; E.C.1.2.1.3) exists as at least three isozymes which are differentially distributed in liver subcellular fractions (designated A2, B4and Cy* for the mitochondrial, soluble and microsomal isozymes respectively) and in various tissues of this animal (Holmes, 1978a; 1978b; Timms&Holmes, 1981). Electrophoretic variants have been previously reported for the A2and B4isozymes among inbred strains of mice, and the genetic loci (designatedAhd‐1andAhd‐2) have been localized on chromosomes 4 and 19 respectively (Holmes, 1978b; Timms&Holmes, 1980). This paper describes further genetic analyses of AHD‐A2enablingAhd‐1to be positioned at the non‐centromeric end of chromosome 4.Forty‐three inbred strains ofMus musculuswere used in these studies (Table 1). Two series of matings were carried out.1) Female SM/J mice and male NZC/B1 mice were mated to obtain F, female offspring which were backcrossed to male NZC/B1 mice. These progeny were used to examine the segregation and linkage relationship ofb(brown),Pgm‐2(encoding phosphoglucomutase B) andAhd‐1(Table 2).2) Female C57BL/6J mice and male SM/J. mice were mated to obtain F, female offspring which were backcrossed to male SM/J mice. The segregation and linkage relationship ofPgm‐2, Gpd‐1(encoding the liver and kidney isozyme of hexose‐6 phosphate dehydrogenase) andAhd‐1were examined for these backcross progeny (Table 3). Methods for preparing liver and kidney extracts and the cellulose acetate electrophoresis procedure for typingAhd‐1, Pgm‐2andGpd‐1have been previously described (Holmes, 1978b).A previous study has described the electrophoretic patterns for allelic variants for mitochondria1 AHD and of the hybrid phenotype for this enzyme (Holmes, 1978b). The three‐allelic isozyme pattern for hybrid animals was consistent with a dimeric subunit structure: AHD‐A1A2, AHD‐A1A2and AHD‐3, with the A1 and A2 subunits being encoded by separate alleles at a single locus, designatedAhd‐1 (Ahd‐1oandAhd‐1brespectively). The distribution of these alleles among 43 inbred strains of mice is given in Table 1. The allelic variants were approximately equally distributed among the inbred strains examined and no divergence of phenotype was observed among the 6 substrains of C57BL mice(Ahd‐1aallele) and 5 substrains of BALB/c(Ahd‐1ballele) mice examined.Genetic variants for phosphoglucomutase‐B (PGM‐B) have been reported by Shows, Ruddle and Roderick (1969) and the gene (Pgm‐2) was subsequently localized on chromosome 4 nearb(brown) by Chapman, Ruddle and Roderick (1970). Table 2 illustrates the results of a three‐point cross betweenb, Pgm‐2andAhd‐1.Variation from the expected 1:1:1:1:1:1 ratio for unlinked loci was significant(x2= 73.15; 7 df; P<1 × 10‐5), indicating that the three loci are linked. Recombination frequency data are consistent with the gene order:b‐Pgm‐2‐Ahd‐1The second cross examined the segregation ofPgm‐2, Ahd‐1andGpd‐1loci (Table 3). The latter locus has been previously positioned on chromosome 4 (linkage group VIII) by Hutton&Roderick (1970) and Chapman (1975), and has been used to localizeAhd‐1in this region(Ahd‐1andGpd‐1exhibit a recombination frequency of 10.3 ± 3.7 %) (Holmes, 1978b). The data from Table 3 is consistent with a gene order ofPgm‐2‐Ahd‐1‐Gpd‐1.The recombination frequency data ofAhd‐1withGpd‐1, Pgm‐2andbalso supports the proposal thatAhd‐1is localized betweenPgm‐2andGpd‐1(Tables 2 and 3; Holmes, 1978b).Recent metabolic studies have indicated that mitochondria1 aldehyde dehydrogenase (AHD) plays a very important role in the metabolism of acetaldehyde derived from ethanol, ensuring a low concentration of acetaldehyde in the blood leaving the liver (Grunnet, 1973; Parilla et al., 1974; Corral1 et al., 1976). Moreover, genetic variation of this isozyme in human livers has been recently reported (Harada et al., 1978), and this polymorphism has been proposed as the molecular basis for individual and racial differences in alcohol sensitivity (Goedde et al., 1979). Consequently, genetic analyses of mitochondria1 AHD are of particular significance to studies on the genetic control of alcohol metabolism in mammals.In summary, this report confirms previous studies which demonstrated that the genetic locus encoding mitochondrial aldehyde dehydrogenase in the mouse(Ahd‐1)is on chromosome 4 (Holmes, 1978b), and positions the gene with respect to b (brown),Pgrn‐2(encoding phosphoglucomutase B) and Gpd‐1(encoding the liver and kidney isozyme of hexose‐6‐phosphate dehydrogenase). In