To solve the problem of whether a common enzyme catalyzes both 5β-cholestane-3α,7α, 12α-triol 27-hydroxylation and 25-hydroxylation of 1α-hydroxyvitamin D3 (a synthetic compound used therapeutically for vitamin D-deficient diseases) in rat liver mitochondria, enzymological and kinetic studies were performed. A cytochrome P-450 was purified from female rat liver mitochondria based on these catalytic activities and it was found that the two enzyme activities accompanied each other at all purification steps. The 5β-cholestane-3α,7α,12α-triol 27-hydroxylation activity of the final preparation had a turnover number of 36min−1, and the value of the corresponding 1α-hydroxyvitamin D3 25-hydroxylation activity was 1.4 min−1. When the enzyme was partially denatured by heating at different temperatures, both enzyme activities declined in a parallel fashion. Treatment of the enzyme with N-bromosuccinimide decreased both enzyme activities in a similar manner. 5β-Cholestane-3α,7α,12α-triol competitively inhibited 25-hydroxylation of 1α-hydroxyvitamin D3 and vice versa. From these results it was concluded that 5β-cholestane-3α,7α, 12α-triol 27-hydroxylation and 1α-hydroxyvitamin D3 25-hydroxylation are catalyzed by a common enzyme in rat liver mitochondria.