Fatty acid compositions of lipids from six bacteria and four yeasts were determined. Fatty acid de novo synthetases were investigated with respect to chain length specificity towards acyl-CoA primers of various chain lengths. Four species of bacteria (Bacillus subtilis, Corynebacterium cyclohexanicum, Micrococcus luteus, and Pseudomonas maltophilia) possess branched-chain fatty acids of the iso and anteiso series as the major acids. De novo synthetases from these organisms exhibited specificity towards the chain length of the primer in the order butyl-CoA > priopionyl-CoA > acetyl-CoA. The remainder, two bacteria and all four yeasts, have the straight-chain type of fatty acids only and fall into two groups: (1) Eschericia coli B, Pseudomonas fluorescens, and Saccharomyces cerevisiae, which utilize the primers in the order acetyl-CoA > priopionyl-CoA > butyryl-CoA; and (2) Candida sake, Candida tropicalis, and Rhodotorula glutinis, which show the order propionyl-CoA > acetyl-CoA > butyryl-CoA. L-alpha-Keto-beta-methylvalerate, a precursor of the branched-chain primers, can be used as a source of primer for fatty acid synthesis by the organisms with branched-chain acids but not by those with the straight-chain type. The results indicate that organisms having straight-chain fatty acids lack the branched-chain equivalents for two reasons: first, their enzymes are not active toward primers with more than three carbons, and second, they lack a system of supply suitable branched-chain primers. It appears that activities of de novo synthetases from the organisms having straight-chain fatty acids generally have much higher activities than those from the organisms possessing branched-chain fatty acids