To examine interactive effects of dietary fat saturation and cholesterol level on serum lipids and de novo cholesterogenesis, moderately hypercholesterolemic subjects were fed solid-foods diets containing 30% fat (80 mg C.1000 Kcal-1) in which 2/3 fat was either corn oil or beef tallow, with and without 120 mg C.1000 Kcal-1, for 5 wk. At the end of each diet period, subjects were given deuterium (D) oxide orally and de novo cholesterol synthesis was measured over 24 h from D incorporation into cholesterol as fractional synthesis rates (FSR) and absolute synthetic rate (ASR) into the rapid exchangeable cholesterol pool. Plasma total and low density lipoprotein levels were elevated (P < 0.01) with both beef tallow and cholesterol feeding. High density lipoprotein (HDL) levels were not influenced by fat saturation or cholesterol level; however, HDL was higher with addition of cholesterol to corn oil versus beef tallow (P < 0.02). Plasma triglycerides were higher (P < 0.02) with beef tallow feeding but were not influenced by cholesterol level. FSR was increased (P < 0.02) by feeding corn oil, versus beef fat, but not by dietary cholesterol level. Calculated cholesterol pools sizes did not differ across groups; however, ASR was also elevated with corn oil versus beef tallow feeding (P < 0.02). Results indicate that corn oil feeding lowers circulating cholesterol by mechanisms other than reduced synthesis, and that cholesterol at the level of supplementation used is not associated with feedback inhibition of cholesterogenesis. However, with the exception of HDL levels, dietary fat saturation and cholesterol levels do not interactively influence circulating lipoprotein cholesterol levels and cholesterol synthesis.