A 190-fold purified preparation of NADP+-specific glutamate dehydrogenase of Salmonella typhimurium was used for the determination of kinetic parameters of the substrates, NADPH, NH4+, and α-ketoglutarate, in the direction of glutamate synthesis, and NADP+ and glutamate in the reverse direction. The kinetic constants determined from this study suggest a biosynthetic role for the enzyme. Based on an analysis of the data derived from initial velocity and product inhibition studies, the reaction mechanism was postulated to be ordered Ter Bi with NADPH as the first substrate to bind in the forward direction, and NADP+ binding first in the reverse direction.Of the several metabolites tested for a possible function in the regulation of GDH activity, only L-malate and L-glutamine appeared to exert an appreciable influence on the enzyme. ATP and AMP at a concentration of 0.8 mM were found to enhance GDH activity by 68% and 6%, respectively, but at high concentrations, both the adenine nucleotides proved to be inhibitory.