A mitochondrial cytochrome P-450 fraction, which catalyzed 25-hydroxylation of vitamin D3 much more efficiently than intact mitochondria was isolated from livers of male and female rats. For comparison, a microsomal cytochrome P-450 fraction was isolated by the same procedures. The mitochondrial cytochrome P-450 from female rats catalyzed 25-hydroxylation as efficiently as the same material from male rats. The microsomal 25-hydroxylation was male specific. The 25-hydroxylase activity in intact mitochondria and the 25-hydroxyvitamin D3 concentration in serum were similar in male and female rats. There was no correlation between the 25-hydroxylase activity in microsomal cytochrome P-450 and the 25-hydroxyvitamin D3 concentration in serum.