Ammonium ions and alanine influence production of the macrolide avermectin in Streptomyces avermitilis. L-Alanine dehydrogenase and alanine aminotransferase are the primary enzymes responsible for regulating the intracellular concentration of alanine and also of ammonium ions. In cultures of S. avermitilis in a chemically defined medium with ammonia or L-alanine as the only nitrogen source, specific activities of both enzymes increased during growth. The alanine dehydrogenase specific activity increased more than 86-fold after the culture was supplemented with 0.2% L-alanine and 5-fold after addition of 0.5% ammonium sulfate, whereas alanine aminotransferase specific activity increased 3- to 4-fold with either substrate. Five isoenzymes of alanine dehydrogenase were detected histochemically in S. avermitilis after native gel electrophoresis. Isoenzyme 1 was induced by alanine and temporarily repressed by high concentrations of ammonium sulfate. The presence of isoenzyme 1 was also related to changes in the kinetic properties of the alanine dehydrogenase reaction measured in crude desarted extracts. A nonlinear double-reciprocal plot was obtained in initial velocity studies using L-alanine as a substrate in the sample induced with L-alanine. The nonlinearity was caused by both substrate inhibition and allosteric regulation (positive cooperativity) by L-alanine. In contrast, the sample induced by ammonium sulfate showed a linear double-reciprocal plot.