The relationship between cell density and de novo synthesis of sterols and fatty acids has been studied in monolayer cultures of L-M cells grown in serum-free medium. Incorporation of radioactivity from [14C] acetate or 3H2O into sterols and fatty acids declined sharply as cultures approached stationary phase. The activities of 3-hydroxy-3-methylglutaryl-CoA reductase and 3-hydroxy-3-methylglutaryl-CoA synthase declined in conjunction with the decrease in sterol synthesis; however, the activity of acetoacetyl-CoA thiolase did not decrease until after sterol synthesis had begun to decline. The magnitude of the initial decline in reductase activity was not diminished when activation of latent enzyme activity was prevented by addition of fluoride to cell homogenates. The diminution in the rate of fatty acid synthesis at high cell density was accompanied by a decrease in the activity of fatty acid synthetase, whereas the activity of acetyl-CoA carboxylase increased slightly. The data suggest that lipogenesis is regulated in coordination with the changes in the rate of cell proliferation that occur when L-M cells attain a high density in monolayer culture. Moreover, these studies establish the feasibility of using the L-M cell culture system to investigate the relationship between cell density and the enzymatic regulation of lipogenesis.