A new model to study cholesterol absorption in the rat intestinal cells is described. Rat intestine epithelial cells IRD98 were incubated with mixed micelles containing bile acid, phospholipid, cholesterol or its nonabsorbable analogue, sitosterol, and trace amounts of [3H]cholesterol or [14C]sitosterol. Cholesterol and sitosterol uptake was then determined following lipid extraction; specific cholesterol uptake was determined as the difference between cholesterol and sitosterol uptake. Cholesterol, but not sitosterol, uptake was time- and dose-dependent and saturable. Loading of cells with non-lipoprotein cholesterol reduced cholesterol, but not sitosterol, uptake in a dose-dependent manner. In contrast, treatment of cells with an inhibitor of cholesterol synthesis, lovastatin, stimulated cholesterol, but not sitosterol, uptake in a dose-dependent manner. Treatment of cells with palmitic, caproic and oleic acids up-regulated specific cholesterol uptake, while linoleic and stearic acids had an opposite effect. None of the fatty acids affected sitosterol uptake.