The effect of saturated and unsaturated fats on in vitro formation and release of lipids and cholesterol from¹⁴C acetate by rat intestinal tissue was investigated. The rats were fed a basal diet enriched with either 25% corn oil or lard and then sacrificed after a 10- or 25-day feeding period. It was observed that a similar¹⁴C lipid content but a greater¹⁴C cholesterol content was found in the intestinal tissue of rats fed corn oil than in rats fed lard for 10 days. After a longer period of feeding of 25 days, the intestinal tissue¹⁴C cholesterol level was decreased in the corn oil fed rats without any significant effect on other lipids. These data suggest that corn oil in some way influences cholesterol biosynthesis depending upon its degree of unsaturation and the period of time for which it is fed. The decrease at the later time might involve some mechanism which aids in getting rid of accumulated tissue cholesterol. Less¹⁴C lipid and¹⁴C cholesterol were released by the intestinal tissue of rats fed the unsaturated fat as compared with those fed the saturated fat, suggesting a possible role in vivo in reducing blood lipids and blood cholesterol levels.