Background and Aims: Cholesterol feeding unexpectedly inhibits cholesterol 7α-hydroxylase in rabbits. The aim of this study was to explore the mechanism. Methods: Twenty male New Zealand white rabbits were fed regular chow with and without 2% cholesterol for 10 days followed by 7 days of bile drainage. The activities of hepatic cholesterol 7α-hydroxylase and sterol 27-hydroxylase that control bile acid synthesis in classic and alternative pathways were related to the size and composition of bile acid pool. Results: After feeding cholesterol, plasma and hepatic cholesterol concentrations increased, the bile acid pool doubled (from 254 ± 44 to 533 ± 51 mg; P < 0.001), cholesterol 7α-hydroxylase activity decreased 68% (P < 0.01), but sterol 27-hydroxylase activity increased 66% (P < 0.05) with increased cholic acid synthesis (P < 0.01). Bile drainage in the cholesterol- fed rabbits depleted the bile acid pool and stimulated down-regulated cholesterol 7α-hydroxylase activity 11.4-fold (P < 0.001), although hepatic cholesterol remained elevated. Hepatic sterol 27-hydroxylase activity was unaffected. Conclusions: Feeding cholesterol increased hepatic cholesterol and stimulated sterol 27-hydroxylase and alternative bile acid synthesis, which expanded the bile acid pool and inhibited cholesterol 7α-hydroxylase in rabbits. In distinction, hepatic sterol 27-hydroxylase was insensitive to changes in the bile acid pool.