Proteins in extracts from cotyledons, hypocotyls, and roots of 5-d-old, dark-grown soybean (Glycine max L. Merr. cv Williams) seedlings were separated by polyacrylamide gel electrophoresis. Three isoforms of glutamate dehydrogenase (CDH) were resolved and visualized i n gels stained for C D H activity. Two isoforms with high electrophoretic mobility, C D H l and CDH2, were in protein extracts from cotyledons and a third isoform with the lowest electrophoretic mobility, CDH3, was identified in protein extracts from root and hypocotyls. Subcellular fractionation of dark-grown soybean tissues demonstrated that CDH3 was associated with intact mitochondria. CDH3 was purified t o homogeneity, as determined by native and sodium dodecyl sulfate-polyacrylamide gels. The isoenzyme was composed of a single 42-kD subunit. The pH optima for the reductive amination and the oxidative deamination reactions were 8.0 and 9.3, respectively. At any given pH, C D H activity was 12-to 50-fold higher in the direction of reductive amination than in the direction of the oxidative deamination reaction. CDH3 had a cofactor preference for NAD(H) over NADP(H). The apparent Michaelis constant values for a-ketoglutarate, ammonium, and NADH at pH 8.0 were 3.6, 35.5, and 0.07 mM, respectively. The apparent Michaelis constant values for glutamate and NAD were 15.8 and 0

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