Despite the remarkable structure of antimycins, their biosynthesis, especially the formation of the rare 3-aminosalicylate moiety, had not been studied in great detail. Here, we report the first details about the formation of this moiety. Candidate genes from Streptomyces ambofaciens for 3-aminosalicylate biosynthesis include a predicted tryptophan-2,3-dioxygenase (SAML0363), a predicted kynureninase (SAML0361), a presumed esterase (SAML0362), a multicomponent oxygenase complex (SAML0365–0369, showing high similarity to the PaaABCDE complex), and an acyl CoA ligase (SAML0371). Feeding studies with stable isotope- and fluorine-labelled precursors (e.g., [2,3,4,5,6,7-2H6]-tryptophan, 15N-anthranilate, 4-fluoroanthranilate, 3-fluoroanthranilate, 6-fluoroanthranilate, N-[13C]formyl-4-fluoroanthranilate, [1-13COOH]anthranilate) revealed that tryptophan is converted to N-formylkynurenine by tryptophan-2,3-dioxygenase, then to anthranilate by kynureninase and esterase. Anthranilate is activated to anthraniloyl-CoA by the acyl CoA ligase, followed by oxidation and an unprecedented 1,2-shift of the carboxylic acid CoA moiety (likely via an epoxide intermediate) catalyzed by the multicomponent oxygenase complex to form 3-aminosalicyloyl-CoA. The 1,2-shift was confirmed by the incorporation patterns of fluoroanthranilates: 4-fluoro and 3-fluoro anthranilates were incorporated into antimycins, while 6-fluoro anthranilate was not. A knock-out mutant of SAML0365–0369 failed to produce antimycins, and complementation with the deleted genes restored production, confirming the role of the multicomponent oxygenase complex. Feeding [1-13COOH]anthranilate showed direct transfer of the carboxyl group to antimycins. In summary, the 3-aminosalicylate moiety of antimycins is derived from tryptophan via anthraniloyl-CoA, involving oxidation and a 1,2-shift of the carboxylic acid CoA moiety.