The lactose content of a semisynthetic diet containing 45% of calories as nonfat milk powder was modified by enzymatic hydrolysis with β-galactosidase or bacterial fermentation with yogurt cultures. Three milk-based diets and a stock diet were fed for 28 days to male rats derived from the Sprague-Dawley strain. Energy consumption, growth rate, feed efficiency, liver weight, liver cholesterol and liver triglyceride concentrations were not significantly different between the four diets. At day 28 serum cholesterol and serum triglyceride concentrations were not different between the milk-based diets. No hypocholesterolemic effect of the milk-based diets was seen at any time compared to the stock diet. Two of four electrophoretic lipoprotein fractions varied with the lactose content of the milk-based diet. The faster alpha lipoprotein decreased while the slower alpha lipoprotein increased with decreasing content of dietary lactose. These data indicate that lactose differs from its constituent monosaccharides, galactose and glucose, in its effect on lipoprotein levels, even though total serum cholesterol and triglycerides do not differ