Phosphoglycerate dehydrogenase (EC 1.1.1.95), an enzyme believed to be involved in the synthesis of serine, an intermediate in ureide biosynthesis, has been purified about 200-fold from nodules of soybean (Glycine max L. Merr. cv Amsoy 71). The reaction was reversible and exhibited a strong pH dependence with optima of 9.4 and 6.1 for the forward and reverse reactions. The K(m) values for the forward reaction were 0.25 millimolar for NAD(+) and 0.29 millimolar for d-3-phosphoglycerate at pH 9.4, while those for the reverse reaction were 12 mum for NADH and 0.15 millimolar for 3-phosphohydroxypyruvate at pH 7.5. NADPH functioned as an alternate reductant with a K(m) of 0.15 millimolar. Product inhibition for the reverse reaction was competitive for NAD(+) with respect to NADH and noncompetitive for phosphoglycerate with respect to phosphohydroxypyruvate. Phosphoglycerate dehydrogenase activity was dependent on inorganic ions and was not inhibited by serine.