Abstract The isozymes of α-glycerophosphate dehydrogenase (α-GPDH) differ markedly with respect to their kinetic and stability parameters. Adult-limited GPDH-1 is stable at 50°C but decays at 57°C, while GPDH-3 is labile at 50°C under similar experimental conditions. By extrapolation of the thermal denaturation curves of crude adult extracts, we estimate GPDH-1 to constitute 76 per cent of the adult α-GPDH activity. Substrate kinetic studies revealed that, at pH 9·5, GPDH-3 exhibits an affinity for α-glycerophosphate which is twofold higher than that of GPDH-1, while Km's for NAD+ are indistinguishable. The apparent Km of GPDH-3 for dihydroxyacetone phosphate is consistently lower than that of GPDH-1 at pH 7·5, whereas at pH 6·7 the latter isozyme's apparent Km approximates that of GPDH-3 at pH 7·5. Indistinguishable molecular weights of 66,000 were estimated by gel filtration for both GPDH-1 and 3. Gene dosage studies indicate that all three α-GPDH isozymes are simultaneously affected by dosage of the Gdh+ locus. These observations support a homomultimeric model of α-GPDH and the isozymes just discussed arise through epigenetic modification of the product of a single structural gene locus.