The nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (l-glutamate:NAD+ oxidoreductase, EC 18.104.22.168) of Chlorella sorokiniana was purified 1,000-fold to electrophoretic homogeneity. The native enzyme was shown to have a molecular weight of 180,000 and to be composed of four identical subunits with a molecular weight of 45,000. The N-terminal amino acid was determined to be lysine. The pH optima for the aminating and deaminating reactions were approximately 8 and 9, respectively. The Km values for α-ketoglutarate, NADH, NH4+, NAD+, and l-glutamate were 2 mm, 0.15 mm, 40 mm, 0.15 mm, and 60 mm, respectively. Whereas the Km for α-ketoglutarate and l-glutamate increased 10-fold, 1 pH unit above or below the pH optima for the aminating or deaminating reactions, respectively, the Km values for NADH and NAD+ were independent of change in pH from 7 to 9.6. By initial velocity, product inhibition, and equilibrium substrate exchange studies, the kinetic mechanism of enzyme was shown to be consistent with a bi uni uni uni ping-pong addition sequence. Although this kinetic mechanism differs from that reported for any other glutamate dehydrogenase, the chemical mechanism still appears to involve the formation of a Schiff base between α-ketoglutarate and an ε-amino group of a lysine residue in the enzyme. The physical, chemical, and kinetic properties of this enzyme differ greatly from those reported for the NH4+-inducible glutamate dehydrogenase in this organism.