Actinomycin synthetase I, which activates the precursor of the chromophore of actinomycin, has been purified nearly 1000-fold from extracts of Streptomyces antibioticus. The enzyme has an M(r) of 45,000 and appears to function as a single polypeptide chain. The formation of the enzyme is repressed when S. antibioticus mycelium is grown on glucose as a carbon source. Several benzoic acid derivatives, including intermediates in the putative pathway for actinomycin biosynthesis, were capable of supporting ATP-pyrophosphate exchange catalyzed by actinomycin synthetase I. Interestingly, three synthetic phenoxaziones also functioned as substrates in the exchange reaction. It proved possible to purify a second enzyme involved in actinomycin biosynthesis, 3-hydroxyanthranilic acid 4-methyltransferase, from the same extracts prepared for the purification of actinomycin synthetase I and by similar procedures. Streptomyces lividans, previously shown to contain a silent gene for the enzyme phenoxazione synthase, apparently does not contain a silent pathway for the complete synthesis of actinomycin since neither actinomycin synthetase I nor the methyltransferase could be detected in strains containing a cloned sequence that activates phenoxazione synthase expression.