—Past studies of atherosclerosis in mice have used chow-based diets supplemented with cholesterol, lipid, and sodium cholate to overcome species resistance to lesion formation. Similar diets have been routinely used in studies with LDL receptor–deficient (LDLR^−/−) mice. The nonphysiological nature and potential toxicity of cholate-containing diets have led to speculation that atherogenesis in these mice may not accurately reflect the human disease process. We have designed a semipurified AIN-76A–based diet that can be fed in powdered, pelleted, or liquid form and manipulated for the precise evaluation of diet–genetic interactions in murine atherosclerosis. LDLR^−/− mice were randomly assigned among 4 diets (n=6/diet) as follows: 1, control, 10% kcal lipid; 2, high fat (40% kcal), moderate cholesterol (0.5% by weight); 3, high fat, high cholesterol (1.25% by weight); and 4, high fat, high cholesterol, and 0.5% (wt/wt) sodium cholate. Fasting serum cholesterol was increased in all cholesterol-supplemented mice compared with controls after 6 or 12 weeks of feeding (P<0.01). The total area of oil red O–stained atherosclerotic lesions was determined from digitally scanned photographs. In contrast to the control group, all mice in cholesterol-supplemented dietary groups 2 to 4 had lesions involving 7.01% to 12.79% area of the thoracic and abdominal aorta at 12 weeks (P<0.002, for each group versus control). The distribution pattern of atherosclerotic lesions was highly reproducible and comparable. The histological features of lesions in mice fed cholate-free or cholate-containing diets were similar. This study shows that sodium cholate is not necessary for the formation of atherosclerosis in LDLR^−/− mice and that precisely defined semipurified diets are a valuable tool for the examination of diet–gene interactions.