AbstractWe evaluated the effect of dietary cholesterol and oxidized cholesterol on the promotion of aberrant crypt foci (ACF), which are putative precancerous lesions in the colon. Sixty female C57BL/6J mice were given four weekly injections (ip) of azoxymethane (AOM) then fed either a control AIN‐76 diet or the control diet supplemented with 0.3% cholesterol or 0.3% oxidized cholesterol for 100 days. The oxidized cholesterol was prepared by heating cholesterol at 110°C for 48 hours. Gas chromatographic analysis of the oxidized cholesterol showed that 96% of the cholesterol was unchanged and less than 2% of the cholesterol was oxidized. The remaining 2% impurities were unidentified and present in both the cholesterol and heated cholesterol. The number of ACF in the group fed cholesterol was significantly lower than the control group (7.9±1.0 vs. 12.5±1.2, p<0.01). The number of ACF in the group fed oxidized cholesterol (10.1±1.1) was not different from the control or cholesterol groups. The size of the ACF (no. of crypts per focus) did not differ between the three dietary groups. Serum low‐density lipoprotein (LDL) cholesterol was greater in the cholesterol‐fed group than the control group (40.5±4.6 vs. 24.3±3.6 mg/dl, p<0.05). LDL cholesterol from the animals fed oxidized cholesterol (37.7 + 4.7 mg/dl) was not different from the control or cholesterol‐fed animals. Total and high‐density lipoprotein (HDL) cholesterol did not differ between the groups. The results show that dietary cholesterol significantly inhibits the promotion of ACF in the colon. The elevated LDL cholesterol may inhibit de novo cholesterol synthesis in the preneoplastic colonic epithelial cells, thereby inhibiting DNA synthesis and cell proliferation.