Lipid synthesis was studied in intestinal mucosal cells isolated from rats fed a high fat or a high sucrose diet. The cells actively incorporated 14C(1)-labeled free fatty acids into glycerolipids [( 1-14C]acetate was utilized for both fatty acid and cholesterol synthesis), while [14C(U)]glucose label was found in cholesterol and in the glycerol moiety of glycerolipids, but not in fatty acids. Sucrose feeding resulted in increased acetate incorporation into cholesterol, but not into fatty acids while the high fat diet markedly depressed the incorporation of acetate. In contrast, fat feeding increased both glucose and fatty acid incorporation into glycerolipids, as well as glucose incorporation into cholesterol. Using the incorporation of glucose into lipid glycerol as an estimate of the phosphatidic acid pathway, it was found that this pathway was stimulated by both fat and carbohydrate feeding. The results suggest that differences in the regulation of cholesterol and glycerolipid synthesis in the intestine compared with adipose tissue and liver may relate to the role of intestine in synthesizing lipoproteins for lipid transport.