The biosynthesis of macbecin, a new antibiotic of the benzenoid ansamycin group produced by Nocardia sp. No. C-14919, was investigated by incorporation of 14C- and 13C-labeled compounds into the antibiotic molecule and analysis of 13C-NMR spectra. Nocardia sp. No. C-14919 was cultured in specific media, with labeled compounds fed into the fermentation medium after 48 hours. Labeled macbecin was extracted, purified, and analyzed for radioactivity and 13C-NMR spectra. Results showed L-[methyl-14C]methionine (44.1%) and [2-14C]propionate (12.1%) were effectively incorporated into macbecin, while acetate, glucose, and L-[carbamoyl-14C]citrulline were also incorporated but at lower rates. Acid hydrolysis of macbecin labeled with L-[carbamoyl-14C]citrulline revealed most radioactivity was in carbon dioxide from the carbamoyl moiety, indicating the C-13 carbamoyl group of macbecin derives from the carbamoyl group of citrulline. 13C-NMR analysis showed [1-13C]propionate enriched C-7, C-11, C-13, C-15, and C-19; [3-13C]propionate enriched methyl carbons at C-8, C-12, C-14, C-16, and C-20; L-[methyl-13C]methionine enriched methoxyl carbons at C-17, C-18, and C-21; and [2-13C]acetate enriched C-10. It is presumed that the main carbon skeleton of the macbecin molecule is derived from acetate, propionate, methionine, and citrulline, with the C-13 carbamoyl moiety from citrulline's carbamoyl group and methoxyl groups from methionine's methyl group.