Activity of glutamine synthetase I (GSI) from Streptomyces aureofaciens increased markedly during tetracycline production phase. The purified GSI exhibited a low affinity for glutamate but high affinities for ATP and ammonium. Its Mn2+-dependent activity was more sensitive to feedback inhibitors than Mg2+-dependent activity. Both the activities were significantly stimulated by Co2+ but inhibited by other divalent cations. ADP was a strong inhibitor. These results suggest that GSI activity is regulated by availability of substrates, feedback inhibitors, divalent cations, and cell energy charge.