Laboratory opossums (Monodelphis domestica) show extreme genetic variability in their responsiveness to dietary lipids; a great proportion of the genetic variability in responsiveness is due to a single major gene. To determine whether the major gene for dietary response detected by genetic analysis in opossums is responsive to dietary cholesterol or dietary saturated fat, or a combination of both, we used males and females of susceptible and resistant lines of laboratory opossums that were 5 to 7 months old. The animals were challenged with three different experimental diets (high-cholesterol diets with or without high saturated fat from lard or coconut oil) and plasma lipoproteins were measured. Plasma and VLDL+LDL-cholesterol concentrations increased several-fold when the animals were fed the diet containing elevated cholesterol (P>0·001) or elevated cholesterol and fat (P>0·001) and differed between the two lines when they were fed high-cholesterol diets with or without fat (P>0·001). Plasma HDL-cholesterol concentrations were higher (P>0·05) in animals of the resistant line than in the susceptible line when they were fed the basal diet (550 (sem 30) v. 440 (sem 20) mg/l) and when they were fed the low-cholesterol and high-fat diet (600 (sem 30) v. 490 (sem 30) mg/l). Dietary coconut oil and lard had similar effects on plasma lipoprotein cholesterol concentrations in the susceptible line of opossums. A reduction in dietary cholesterol by 50% with either the lard or coconut oil blunted the plasma cholesterol response. The results from the present studies suggest that the major gene for dietary response previously detected by genetic analysis in laboratory opossums affects the response to dietary cholesterol but not to saturated fat.