NADP(+)-dependent serine dehydrogenase [EC 1.1.1.-], which catalyzes the oxidation of the hydroxyl group of serine to form 2-aminomalonate semialdehyde, was purified to homogeneity from a crude extract of Agrobacterium tumefaciens ICR 1600. The enzyme had a molecular mass of about 100 kDa and consisted of four identical subunits. In addition to L-serine, D-serine, L-glycerate, D-glycerate, and 2-methyl-DL-serine were substrates. However, O-methyl-DL-serine and L-threonine were inert. The enzyme showed maximal activity at about pH 9 for the oxidation of L-serine. The enzyme required NADP+ as a coenzyme, NAD+ was inert. The enzyme was not inhibited by EDTA, o-phenanthroline, or alpha,alpha'-dipyridyl, but was inhibited by HgCl2, p-chloromercuribenzoate, L-cysteine, D-cysteine, malonate, 2-methylmalonate, and tartronate. The Michaelis constants for L-serine, D-serine, and NADP+ were 42, 44, and 0.029 mM, respectively.