After growth of Mycobacterium vaccae strain JOB5 on acetate or propane, the cellular fatty acids were isolated and identified by a combination of gas-chromatographic, mass-spectral, and chemical means. The fatty acids ranged from C12 to C19 and were a mixture of saturated, monounsaturated, and methyl-branched components. The double bond was in the delta9 position in the C15 to C18 unsaturated acids. The single methyl branch was located on the C10 position of Br-C17, Br-C18, and Br-C19 fatty acids. Branched-chain synthesis occurs at the expense of an unsaturated precursor fatty acid; the double bond serves as the site of methylation. Results suggest that S-adenosylmethionine is the methyl donor involved.