We reported previously that dietary cholesterol produces hypertriglyceridemia in the rat, accompanied by reduced oxidation and increased incorporation of exogenous fatty acid into hepatic triglyceride and increased secretion of very low density lipoprotein. We now report that dietary cholesterol also increases net hepatic fatty acid synthesis and the incorporation of newly synthesized fatty acid into hepatic triglyceride in vivo. Male rats were fed a cholesterol-free, semisynthetic diet (5% [w/w] corn oil) for 7 days, or the same diet supplemented with 0.5% cholesterol. On the day of the experiments, fed animals received 5 mCi 3H2O intraperitoneally (i.p.) either at 1200 h (6 h into the light cycle) or at 2400 h (6 h into the dark cycle). Animals were killed 1 h after receiving the radioisotope. Feeding cholesterol increased hepatic triglyceride and cholesteryl ester concentrations, moderately elevated the content of free cholesterol, but did not affect phospholipid levels. Increased net synthesis of fatty acids by livers of animals receiving cholesterol was observed during the dark period; a similar increase during the light period was also observed for incorporation of newly synthesized fatty acid into hepatic phospholipid and cholesteryl ester, although incorporation into triglyceride was of borderline significance (P < 0.06). In other experiments male rats were fed similar diets for 3, 7, or 21 days. Fed animals received 10 mCi 3H2O, i.p. (900-1000 h), and were killed 24 h later. Duration of feeding did not influence rates of net fatty acid synthesis or the stimulation by cholesterol of incorporation of newly synthesized fatty acid into hepatic triglyceride and cholesteryl ester.(ABSTRACT TRUNCATED AT 250 WORDS)