The specific effects of soybean protein on lipid metabolism were determined with highly purified soybean protein. At 5 wk of age, growing rats were fed diets containing 20% highly purified soybean protein or casein supplemented or not with 0.1% cholesterol for 2 mo. Plasma and liver lipid composition, fecal steroid excretion and several hepatic enzyme activities were measured. There were no significant dietary protein-related differences in plasma and liver cholesterol concentrations. When diets were cholesterol free, highly purified soybean protein stimulated fecal neutral and acidic steroid excretion associated with concomitantly higher hydroxy methylglutaryl CoA (HMG-CoA) reductase activity, but lower cholesterol 7alpha-hydroxylase activity. Soybean protein lowered the linoleate desaturation index [20:4(n-6)/18:2(n-6)] in liver microsomal lipids and phospholipids. This may have been due to the reduced microsomal Delta6(n-6) desaturase activity in rats fed soybean protein, whereas Delta5(n-6) desaturase activity did not differ between groups fed the two proteins. Cholesterol supplementation (0.1%) did not affect plasma cholesterol but increased liver cholesterol and triacylglycerol concentrations and reduced HMG-CoA reductase activity; this latter effect was greatest in rats fed soybean protein. Cholesterol 7alpha-hydroxylase activity, however, was diminished only in rats fed casein. Desaturase activities, and particularly Delta5(n-6) activity, were lowered by cholesterol supplementation in rats fed both protein diets, including a significantly lower 20:4(n-6)/18:2(n-6) ratio in liver microsomal lipids and liver phospholipids. Thus although dietary proteins have no effect on serum cholesterol in rats, they affect enzyme activities involved in cholesterol metabolism and fatty acid desaturation.