Since the small intestine contributes significantly to serum cholesterol and very low density lipoprotein levels, acute regulation of lipid synthesis was investigated in isolated rat intestinal cells incubated in Krebs-Ringer bicarbonate buffer with 5 mM glucose and [14c]acetate or 3H2O. Incorporation of [14c]acetate into cellular lipids was 6- to 8-fold greater in crypt than in villus cells. In both cell types the distribution of 14C among the various lipid classes was as follows: 52.5% in triglycerides, diglycerides, and monoglycerides; 22.3% in cholesterol; 8.3% in cholesteryl esters; 1.9% in fatty acids; and 15.0% in phospholidpids. In contrast, the medium lipids contained significantly higher amounts of tri-, di- and monoglycerides (61.1%) and lower amounts of cholesteryl esters (2.3%) and phospholipids (11.9%). After saponification, 2/3 of the recovered 3H2O was in fatty acids and 1/3 in cholesterol. Ethanol (10 mM) tripled 3H2O incorporation into cellular lipids but had no effect on [14c]acetate incorporation. Epinephrine and norepinephrine (10 micron), glucagon (10 micron), dibutyryl cyclic AMP (1MM), dexamethasone (1 mM and 1 micron), and cholera toxin (1 microgram/ml) did not affect [14c]acetate incorporation. We concluded that ehtanol stimulates intestinal lipid synthesis; however, in sharp contrast to their inhibition of lipid synthesis in hepatocytes and adipocytes, catecholamines, glucagon, and dibutyryl cyclic AMP do not inhibit lipid synthesis in intestinal cells.