Physicochemical studies of chicken liver fatty acid synthetase have shown that the enzyme has been isolated in a high state of purity. Polyacrylamide gel electrophoresis of the enzyme preparations in SDS-phosphate, SDS-Tris-glycine, and Tris-glycine buffers indicate that 90% of the protein consists of one component. We have demonstrated that proteolysis of the synthetase may occur during the isolation of the enzyme. In addition, the purified enzyme undergoes proteolysis in the presence of denaturing agents, as is evident from the appearance of numerous bands on SDS gels. The proteolysis may be avoided by rapid purification of the enzyme and by heat treatment of the synthetase in the presence of denaturing agents. In addition, the nutritional state of the animal at the time of sacrifice may affect the banding pattern of the enzyme on SDS gels. Preparations obtained from starved-refed chickens generally gave over 90% of the stain in one band, while preparations obtained from starved animals exhibited several bands. The molecular weight of the native enzyme is 460,000–500,000, while in the presence of Tris-glycine buffer the enzyme rapidly dissociated into subunits of 230,000–250,000 molecular weight. The molecular weight has been estimated by sedimentation equilibrium in the presence of 6 m guanidinium chloride (214,000) and by SDS-polyacrylamide gel electrophoresis (220,000). It is proposed that the fatty acid synthetase consists of two subunits of the same or nearly equal size and, since the subunits contain the catalytic sites and the 4′-phosphopantetheine, the synthesis is a multifunctional enzyme. The amino acid analysis and the 4′-phosphopantetheine content have been determined. As reported earlier (Arslanian, Stoops, Oh, and Wakil, 1976, J. Biol. Chem.251, 3194–3196), the synthetase contains 1.6 mol of 4′-phosphopantetheine/mol of enzyme. In this study, the prosthetic group content was shown to be independent of the procedure used to isolate the 4′-phosphopantetheine for analysis.