The fatty acid pattern in Mycobacterium vaccae strain JOB5 was examined after growth on n-alkanes (C14–C18), 1-alkenes (C14–C18), 2- or3-methyl octadecane, and 8-heptadecene. It was evident that monoterminal oxidation of n-alkanes was followed by β-oxidation and that both parent fatty acid and products of β-oxidation were incorporated into cellular lipids. Radioactive experiments demonstrated that there was desaturation of long-chain fatty acids. There was no evidence of chain elongation. Growth on 1-alkenes resulted in the incorporation of fatty acids that were products of two primary modes of oxidation: (1) methyl group attack resulting in ω-unsaturated fatty acids and (2) double-bond attack resulting in the removal of one carbon from the substrate. Cells of strain JOB5 grown on 2- and 3-methyl octadecane contained the corresponding iso- or anteiso-fatty acids in significant quantity. Cells cultured on 8-heptadecene contained 8- and 9-heptadecenoic acids, 6- and 7-pentadecenoic acids, 9- and 10-methyl hep-tadecanoic acids, and 7- and 8-methyl pentadecanoic acids. Fatty acid composition (C13 to C19) was affected by substrate chain length and was additionally modified by cellular control mechanisms.