Human tissues contain two isozymes of neutral a-glucosidase, neutral a-glucosidase AB and neutral a-glucosidase C (a-D-glucoside glucohydrolase, EC 3.2.1.20). The two isozymes, initially defined on the basis of differences in electrophoretic mobility in starch gel, have also been shown to have other distinguishing biochemical characteristics including different substrate specificites. Rodent tissues contain apparently homologous isozymes of neutral a-glucosidase. The mouse and human a-glucosidase C isozymes, but not the AB isozyme(s), can be distinguished by the difference in their electrophoretic mobility. This difference has previously enabled us to use human-mouse somatic cell hybrids to assign the structural gene for human a-glucosidase C to chromosome 15. We now report the differentiation of mouse and human neutral a-glucosidase AB isozymes by rocket immunoelectrophoresis, using an antibody raised in mice against purified human placental neutral a-glucosidase AB. This antibody precipitated both the A and B bands of human neutral a-glucosidase AB and did not cross react with mouse enzyme as determined by Ouchterlony double immunodiffusion and by rocket immunoelectrophoresis. Using this antibody, the segregation of human neutral a-glucosidase AB was examined in 41 mouse x human hybrid clones. Thirty-eight hybrid clones, derived from fusions of RAG x seven different human cells, showed 100% concordant segregation of human neutral a-glucosidase AB and the 11. Three additional clones, derived from a fusion of tetraploid murine erythroleukemia cells (2S-MEL) x diploid human fibroblasts carrying a translocation chromosome(s) allowed the regional localization of the gene to the long arm of 11 (11q13→11qter