The enzyme previously considered as an isozyme (E4, ALDH IV) of human liver aldehyde dehydrogenase (NAD+) (EC 1.2.1.3) has been purified to homogeneity by the use of ion exchange chromatography on CM-Sephadex and affinity chromatography on Blue Sepharose CL-6B and 5'-AMP Sepharose 4B and identified as glutamic gamma-semialdehyde dehydrogenase, or more precisely 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12). Glutamic gamma-semialdehyde dehydrogenase was never previously purified to homogeneity from any mammalian species. The homogeneous enzyme is seen on isoelectric focusing gels as two fine bands separated by 0.12 pH units: pI = 6.89 and 6.77. In addition, the enzyme also appears as two bands in gradient gels; however, in polyacrylamide gels containing sodium dodecyl sulfate the enzyme migrates as one band, indicating that its subunits are of identical size. Because the enzyme molecule is considerably smaller (Mr approximately 142,000-170,000) than that of aldehyde dehydrogenases (EC 1.2.1.3) (Greenfield, N. J., and Pietruszko, R. (1977) Biochim. Biophys. Acta 483, 35-45; Mr approximately 220,000) and its subunit weight is different (70,600 versus approximately 54,000 for E1 and E2 isozymes), the enzyme is not an isozyme of aldehyde dehydrogenase previously described. The Michaelis constants for glutamic gamma-semialdehyde dehydrogenase with acetaldehyde and propionaldehyde are in the millimolar range. Its substrate specificity within the straight chain aliphatic aldehyde series is essentially confined to that of acetaldehyde and propionaldehyde with butyraldehyde and longer chain length aldehydes being considerably less active. Other substrates include succinic, glutaric, and adipic semialdehydes in addition to glutamic gamma-semialdehyde. The reaction velocity with glutamic gamma-semialdehyde is at least an order of magnitude larger than with carboxylic acid semialdehydes. Aspartic beta-semialdehyde is not a substrate. The reaction catalyzed appears to be irreversible. Although NADP can be used, NAD is the preferred coenzyme. The enzyme also exhibits an unusual property of being subject to substrate inhibition by NAD.