Biosynthetic studies on esperamicin Al (esp At) were carried out by examining the incorporation of singly and doubly C-13-labeled acetates, L-[methyl-C-13]methionine and (Na2SO4)-S-34, by cultures of Actinomadura verrucosospora MU-5019. The acetate incorporation results show that the C-15 bicyclic enediyne core of esp A1 is derived from head-to-tail condensation of seven acetate units and the uncoupled carbon attached to the trisulfide unit is derived from the C2 of acetate. The L-[methyl-C-13]methionine incorporation result shows that the S-methyl groups of the trisulfide and the thiosugar and the O-methyl groups of the aminosugar, the aromatic chromophore, and the carbamate moiety are derived from L-methionine via S-adenosylmethionine. Using (Na2SO4)-S-34 as the sole sulfur source in the fermentation and by mass spectrometric analysis, we have demonstrated that all four sulfur atoms in esperamicin A1c (esp A1c) can be derived from (Na2SO4)-S-34. On the basis of the C-13-labeled acetate-enrichment pattern, the enediyne ring moiety of esp A1 maybe derived from an octaketide with the loss of the Cl of the end acetate unit. The acetate-enrichment pattern of the enediyne moiety of esp A1 is in good agreement with that of dynemicin A (DNM-A). The two carbons comprising the yne moieties of esp A1 and DNM-A are derived from separate acetate units. The corresponding carbons in chromophore A of neocarzinostatin (NCS Chrom A) are derived from the same acetate units. This may suggest that enediyne cores of esp A1 and DNM-A are biosynthesized from a common precursor while NCS Chrom A is biosynthesized via a different process.