Feeding of [1,2-I3C,]acetate to cultures of Streptomyces muruyumaensis gave kinamycin C and kinamycin D, each showing intact incorporation of acetate at carbons 1/2, 3/4, 10/5, 6/7, 8/9, 6'/1', 2'/CH3, and 4'/5' and at the esters, as well as simple enrichment in C-3', as shown by I3C NMR analysis. The orientation of each acetate in kinamycin D was revealed by incorporation of [1-13C,2,2,2-2H3]acetate. I3C NMR analysis indicated enrichment at carbons 1, 3, 6, 8, 10, 2', 4', and 6' and the ester carbonyls; isotope-shifted resonances were also observed for carbons 6, 10, and 2' and the ester carbonyls, indicating retention of deuterium at the adjacent positions from the original acetate. Thus, the biosynthetic origin of all the carbons of kinamycin, except the cyano moiety, was revealed. When [ 1-w~ ,l-1802]acetate was fed, isotope-shifted peaks were observed for carbons 1, 8, and 4' and the ester carbonyls of kinamycin C and of kinamycin D, indicating retention of I80 at these positions, and when kinamycin D was produced in the presence of '*02, isotope-shifted peaks were observed for carbons 4, l', and 2'. These results are consistent with the kinamycin skeleton being derived from acetate via 1,3,8-trihydroxynaphthalene-subsequently oxidized either to 2-hydroxyjuglone or to juglone-and 4-amino-2-hydroxytoluic acid. The D ring is most reasonably generated by oxidation of tetracyclic intermediate 18b to hydroquinone 19b and then to epoxide 2Ob, followed by rearrangement to epoxyquinol 21, reduction, and epoxide opening with trans attack by water.