The product distribution of Brevibacterium ammoniagenes fatty acid synthetase has been investigated using propionyl-CoA instead of acetyl-CoA as the primer. The synthetase produces not only an odd-numbered fatty acid (heptadecanoic acid) but also even-numbered fatty acids (stearic and oleic acids) in the presence of propionyl-CoA. The amounts of heptadecanoic, stearic and oleic acids increased with increasing concentration of propionyl-CoA. However, the formation of heptadecenoic acid (C_17:1) was not observed under any conditions tested. The failure of C_17:1 synthesis suggested that the enzyme component catalyzing the β, γ-dehydration, which is responsible for the synthesis of unsaturated fatty acids, has a high degree of chain length specificity. Under standard assay conditions, stearic acid predominated and heptadecanoic and oleic acids were found in lesser amounts. Mass spectrometric analyses of fatty acids synthesized either from [²H] propionyl-CoA or in ²H₂O revealed that propionyl-CoA is utilized as the priming substrate for the synthesis of heptadecanoic acid and that an acetyl residues, which is formed by the decarboxylation of malonyl-CoA, served as the priming substrate for the syntheses of stearic and oleic acids. No evidence was obtained for the direct decarboxylation of malonyl-CoA to acetyl-CoA in this reaction. It is concluded that the decarboxylation of the malonyl moiety bound to the synthetase occurs efficiently only in the course of fatty acid synthesis. A hypothetical scheme is presented to explain the propionyl-CoA-dependent decarboxylation of the malonyl moiety.