Mevinolin (lovastatin), a competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase, directly inhibited acyl-CoA cholesteryl acyltransferase in rabbit intestinal microsomes at a dose of 20 micrograms/ml or more. Lineweaver-Burk analysis showed a competitive type of inhibition with respect to oleoyl-CoA. In cultured intestinal Caco-2 cells, mevinolin reduced [14C]oleate incorporation into cholesteryl-esters by 86% of controls at doses as low as 0.1 micrograms/ml. However, in cells whose activity of acyl-CoA cholesteryl acyltransferase was stimulated 7-fold by 10 mM mevalonolactone, a significant inhibitory effect on cholesteryl-ester formation could not be detected, even at 40 micrograms/ml of mevinolin. In contrast, cells supplied with liposomal cholesterol or cholesterol derived from low-density lipoproteins showed a marked reduction of cholesteryl-ester formation in the presence of 10 or 0.1 micrograms/ml of mevinolin, respectively. It is concluded that the observed suppressive effects of mevinolin on cholesterol esterification in cultured Caco-2 cells are indirect and possibly caused by changes in the acyl-CoA cholesteryl acyltransferase substrate pool or intracellular cholesterol transport.