The regulation of cholesterol synthesis has been studied using a rat epithelial intestinal cell line (IRD 98) as a cellular model. As observed in other cell types, mevinolin increases the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) and concomitantly reduces the incorporation of [14C]acetate into cholesterol. Free cholesterol is able to suppress reductase activity. In contrast, when cells are shifted from standard culture medium to lipoprotein-deficient medium, an increase in hydroxymethylglutaryl-CoA reductase specific activity (2-5-fold) is observed. The possible regulatory roles of the different classes of human lipoproteins were thus compared. The effects of a long-term exposure to LDL and HDL vary according to cell density. In actively growing cells, VLDL and LDL cause a decrease in the level of hydroxymethylglutaryl-CoA reductase, whereas HDL do not have a significant effect. In contrast, in subconfluent preresting cells, HDL provoke large decreases in hydroxymethylglutaryl-CoA reductase activity as compared to VLDL and LDL. While LDL binding is constant, the maximal binding capacity of HDL in subconfluent cells is seven times that of actively growing cells. Altogether, these results suggest an important role for HDL in the regulation of intestinal cholesterol synthesis.