Caco-2 cells grown on membrane filters were used as a model to study the effects of NB-598, an inhibitor of squalene epoxidase, on cholesterol absorption from the intestinal epithelia. NB-598 (10 μM) inhibited the synthesis of sterol and sterol ester from [ 14C]acetate without affecting the synthesis of other lipids such as phospholipids (PL), free fatty acids (FFA) and triacylglycerol (TG). When labeled lipid was apically loaded as a micellar lipid solution into Caco-2 cell cultures, NB-598 reduced basolaterally secreted radioactivity in cholesterol, cholesterol ester, PL and TG. Furthermore, NB-598 suppressed the basolateral secretion of apolipoprotein (apo) B. When microsomes prepared from control Caco-2 cells were incubated with 10 μM NB-598, acyl CoA:cholesterol acyltransferase (ACAT) activity was inhibited slightly. After incubating Caco-2 cells with 10 μM NB-598, a slight reduction in cellular ACAT activity was also observed. These results suggest that suppression of the secretion of particles containing apo B and reduction of cellular ACAT activity in the intestinal epithelia are part of the mechanism of the cholesterol-lowering effect of NB-598.