Biosynthetic studies on neocarzinostatin chromophore A (NCS Chrom A) were carried out on the basis of the incorporation of singly and doubly ¹³C labeled acetate precursors as well as radiolabeled [methyl-³H]methionine, [¹⁴C]sodium bicarbonate, and [¹⁴C]sodium acetate by cultures of Streptomyces carzinostaticus (ATCC # 15944 F-42). The results suggest that the N-methyl of the fucosamine and the 0-methyl of the naphthoic acid moieties are derived from methionine via S-adenosylmethionine and the cyclic carbonate carbonyl carbon from carbonate. The acetate incorporation results show that the C₁₂ naphthoic acid ring is derived from a hexaketide. The intriguing C₁₄ cyclic carbonate/bicyclo[7.3.0]dodecadienediyne ring system, on the other hand, appears to be derived from a minimum of eight head to tail coupled acetate units which is discussed in terms of the oleate-crepenynate biosynthetic pathway for polyacetylenes. The related C₁₅ enediyne ring skeleton in the esperamicin/calicheamicin class of antitumor antibiotics may be similarly derived. These incorporation experiments provide independent support for the unprecedented structure of NCS Chrom A.