L-Alanine dehydrogenase was found in extracts of the antibiotic producer Streptomyces clavuligerus. The enzyme was induced by ammonia, and the level of induction was dependent on the extracellular concentraction. L-Alanine was the only amino acid able to induce alanine dehydrogenase. The enzyme was characterized from a 38-fold purified preparation. Pyruvate (Km = 1.1 mM), ammonia (Km = 20 mM) and NADH (Km = 0.14 mM) were required for the reductive amination, and L-alanine (Km = 9.1 mM) and NAD (Km = 0.5 mM) for the oxidative deaminating reaction. The aminating reaction was inhibited by alanine, serine and NADPH. Alanine inhibited uncompetitively with respect to NADH (Ki = 1.6 mM) and noncompetitively with respect to ammonia (Ki = 2.0 mM) and pyruvate (Ki = 3.0 mM). In the aminating reaction 3-hydroxypyruvate, glyoxylate and 2-oxobutyrate could partially (6--7%) substitute pyruvate. Alanine dehydrogenase from S. clavuligerus differed with respect to its molecular weight (92000) and its kinetic properties from those described for other microorganisms.