Fecal bile acid and neutral sterol excretion rates were determined in five healthy young men when serum cholesterol changes were induced by isocaloric substitution of an unsaturated (safflower oil) for a saturated fat (butter). The isotope balance method was used after the intravenous injection of cholesterol-4-(14)C. A feces extraction method is presented which permits essentially complete separation of fecal neutral sterols and bile acids. There was a significant increase in the total excretion of the fecal end products of cholesterol metabolism from 966 +/- 42 mg/day on saturated fat to 1147 +/- 45 mg/day on unsaturated fat, and the increase was equally distributed between the neutral sterol and bile acid fractions. With the substitution of dietary fats, regardless of the sequence of their feeding, there was a 28% reduction in serum cholesterol concentration during ingestion of the unsaturated fat. There were reciprocal changes in serum cholesterol levels and fecal steroid excretion with the substitution of one type of fat for the other. The changes in plasma cholesterol content were more than adequately balanced by the reciprocal changes in fecal cholesterol end product excretion. The findings in this study agree with several previous reports in supporting the hypothesis that the hypocholesteremic action of dietary unsaturated fatty acids is associated with an increase in the fecal loss of bile acids and neutral sterols.