When propionyl-CoA was substituted for either acetyl-CoA or butyryl-CoA in the presence of [14C]malonyl-CoA and NADPH, the pure human liver fatty acid synthetase complex synthesized only straight-chain, saturated, 15- and 17-carbon radioactive fatty acids. At optimal concentrations, propionyl-CoA was a better primer of fatty acid synthesis than acetyl-CoA. Methylmalonyl-CoA inhibited the synthetase competitively with respect to malonyl-CoA. TheKiwas calculated to be 8.4 μM. These findings provide anin vitromodel and offer a direct explanation at the molecular level for some of the abnormal manifestations observed in diseases characterized by increased cellular concentrations of propionyl-CoA and methylmalonyl-CoA.