A novel ansamycin, rifamycin W, was isolated from a mutant strain of Nocardia mediterranei. The metabolic origin of rifamycin W was studied by (13)C nuclear magnetic resonance spectroscopy. Examination of the proton-decoupled pulse and Fourier transform (13)C spectra of rifamycin W biogenetically enriched with [1-(13)C]-, [2-(13)C]-, and [3-(13)C]propionate and with [1-(13)C]acetate has revealed that the alignment of acetate and propionate units corresponds to that previously proposed for rifamycin S. Washed mycelium from a rifamycin B-producing strain of N. mediterranei transformed rifamycin W into rifamycin B. We suggest that rifamycin W is a normal intermediate in the biosynthesis of the other rifamycins. These results, together with the structural similarity of rifamycin W to the streptovaricins, reinforce our hypothesis that a common progenitor is involved in the biogenesis of all naphthalenic ansamycins.