SummaryTo explore further the factors that underly the deficient generation of 3,5,3′‐triiodothyronine (T3) from thyroxine (T4) (T3‐neogenesis) in fetal liver, studies were performed in homogenates and subcellular fractions of liver from pregnant rabbits and their corresponding fetuses. In whole homogenates, T3‐neogenesis (percentage added T4:50 mg protein) during 3 hr incubations of specimens from fetuses proceeded at a much slower rate (1.7 ± 0.5, mean ± S.D.) than in specimens from the corresponding does (1.4 ± 5.3). Dithiothreitol (DTT, 20 mM), a known stimulant of T3‐neogenesis, did not significantly alter the activity of fetal specimens (2.6 ± 1.3), but significantly increased activity in those obtained from does (18.2 ± 7.4). To investigate the basis for these differences, additional experiments were performed in which microsomal and cytosolic fractions from fetal and maternal livers were variously mixed and T3‐neogenesis was assessed in the presence and absence of DTT. Activity of fetal microsomes was consistently less than in maternal microsomes, regardless of the source of the cytosols with which they were incubated. Further, mixtures containing fetal microsomes were not stimulated by DTT, whereas those containing maternal microsomes were. In addition to this evidence of an abnormality with respect to the fetal enzyme, evidence was obtained of a concomitant abnormality in fetal cytosol fractions. Thus, T3‐neogenesis by maternal microsomes was consistently greater when incubated with maternal cytosol than with fetal cytosol, presumably owing to lesser support of T3‐neogenesis by cofactors known to be present in cytosol. Additional evidence of an abnormality in the microsomal enzyme of the fetus was obtained in studies of the kinetics of T3‐neogenesis. In the presence of DTT, derived values for the Kmof the fetal enzyme were approximately 10‐fold higher than were those for the maternal enzyme (5.9 ×−5and 7.1 × 10−6M).The studies suggest that the defective generation of T3from T4seen in fetal liver from other species is also present in the liver of the fetal rabbit. Lower activity in the fetal liver is due in part to a lesser activity of cytosolic cofactors, but mainly to an abnormality in the T3‐generating enzyme, reflecting either a lower concentration of enzyme, a lesser responsiveness to stimulatory cofactors, or a combination of the two.SpeculationThe enzyme in fetal liver that converts thyroxine to 3,5,3′‐triiodothyronine behaves differently from that in maternal liver with respect to its affinity for substrate and its response to stimulatory sulfhydryl cofactor. Fetal enzyme may be an isozymic variant of the adult enzyme, as has been reported for other hepatic enzymes. The relation between fetal and maternal enzymes may be analogous to that which exists with respect to enzymes of normal and malignant tissue.