Plasma 7α-hydroxy-4-cholesten-3-one has been used as an index of hepatic bile acid synthesis. The aim of the current study was to ascertain whether the level of this oxysterol reflects hepatic cholesterol 7α-hydroxylase activity when plasma cholesterol concentrations are markedly changed. In addition, the relationship of hepatic sterol 27-hydroxylase activity with plasma concentrations of 27-hydroxycholesterol and 3β-hydroxy-5-cholestenoic acid was studied. We used New Zealand white rabbits fed 2% cholesterol for 5 or 10 days and/or constructed bile fistula. Feeding cholesterol markedly increased and bile drainage reduced plasma cholesterol concentrations. Initially, in these models there was no correlation between plasma 7α-hydroxy-4-cholesten-3-one concentrations and hepatic cholesterol 7α-hydroxylase activities ( r = −0.24, n = 10). Cholesterol feeding was associated with downregulated 7α-hydroxylase activities, while plasma 7α-hydroxy-4-cholesten-3-one concentrations were elevated in the presence of increased plasma cholesterol levels. However, this discrepancy was overcome and significant correlation was observed ( r = 0.73, P < .05, n = 10) by expressing 7α-hydroxy-4-cholesten-3-one levels relative to cholesterol. In contrast, hepatic sterol 27-hydroxylase activities were not significantly correlated with plasma absolute ( r = 0.23, difference not significant NS, n = 10) nor cholesterol-related levels of 27-hydroxycholesterol ( r = −0.13, NS, n = 10), or 3β-hydroxy-5-cholestenoic acid concentrations ( r = 0.30, NS, n = 10). In conclusion, plasma 7α-hydroxy-4-cholesten-3-one concentrations reflected hepatic cholesterol 7α-hydroxylase activities when the sterol levels were adjusted to plasma cholesterol concentrations in rabbits with hypercholesterolemia. The results suggest that plasma 7α-hydroxy-4-cholesten-3-one relative to cholesterol is a better marker for hepatic cholesterol 7α-hydroxylase activity than the absolute concentration when hypercholesterolemia is present. In contrast, 27-hydroxycholesterol and 3β-hydroxy-5-cholestenoic acid levels in plasma did not reflect hepatic sterol 27-hydroxylase activities even if the levels were adjusted to plasma cholesterol concentrations.