The role of liver acyl-CoA:cholesterol acyltransferase 2 (ACAT2), earlier shown to be the principal ACAT enzyme within primate hepatocytes, as a regulator of the hypercholesterolemia induced by dietary cholesterol was studied. At the end of low and high cholesterol diet periods, liver biopsies were taken from cynomolgus monkeys, a species highly responsive to dietary cholesterol, and less responsive African green monkeys. Liver cholesterol and cholesteryl ester concentrations were highest in cynomolgus monkeys fed cholesterol, despite the fact that in order to induce equivalent hypercholesterolemia, dietary cholesterol levels were 50% lower than was fed to green monkeys. Hepatic cholesteryl oleate secretion rate, measured during liver perfusion as an indicator of ACAT activity, was significantly higher in cynomolgus monkeys. Liver microsomal ACAT activity was 2-3-fold higher in cynomolgus monkeys than in green monkeys. The responses of ACAT2 were compared with those of ACAT1 that is found primarily in Kupffer cells. ACAT2 protein mass was significantly correlated to microsomal total ACAT activity in both species; ACAT1 mass was less well correlated. Dietary cholesterol induced a significant 3-fold increase of ACAT2 protein mass in cynomolgus monkeys, a much greater increase than was found for mRNA abundance; neither ACAT2 mRNA nor protein was diet-responsive in green monkeys. In cynomolgus monkeys but not in green monkeys, liver free cholesterol concentrations were elevated when cholesterol was fed and were correlated with ACAT2 protein levels. The data suggest a mechanism whereby the elevation of hepatic free cholesterol concentrations by dietary cholesterol, seen only in cynomolgus monkeys, resulted in higher ACAT2 protein levels in hepatocytes, either through increased production or stabilization of the protein. Regulation of ACAT2 gene transcription was not a factor.