Lipid synthesis from acetate and glucose has been studied in fibroblast cultures grown in lipid free medium containing various concentrations of acetate and glucose. Their role in lipid synthesis was evaluated by comparing the specific activities of total cell lipid, and sterol and fatty acid subfractions, to that of the carbon source in the medium. When glucose was present at 1000 microgram/ml (5.6 mM) and acetate at 100 microgram/ml (1.2 mM), the majority of the total cell lipid and subfractions were derived from glucose. As acetate concentrations increased, lipid synthesis from acetate increased, and glucose declined as a carbon source; in cells grown in the presence of 1000 microgram/ml (12 mM) acetate more of the lipid was derived from acetate than from glucose. When acetate was present at 1000 microgram/ml (12 mM) and glucose was varied from 1000 to 200 microgram/ml (5.6 to 1.1 mM), the relative amount of lipid synthesis from acetate was further increased. The data indicate that acetate can serve as a significant carbon source for lipid synthesis in cultured fibroblasts when it is presented to the cells at high extracellular concentrations relative to glucose.