A major action of estradiol in the corpus luteum of the pregnant rat is to increase the supply of cholesterol substrate for progesterone production by stimulating both cholesterol synthesis and uptake. To determine whether this steroid also affects cholesterol metabolism and transport, estradiol's action on the expression of cytochrome P450 side-chain cleavage enzyme (P450scc) and the cholesterol transport protein, sterol carrier protein-2 (SCP2), was examined. Mitochondria isolated from corpora lutea of estradiol-treated rats secreted significantly more progestagen than mitochondria of control corpora lutea. Several findings indicate that estradiol enhances cholesterol transport and availability to the P450scc rather than affects the expression of this enzyme: 1) the difference in mitochondrial progestagen synthesis induced by estradiol was obliterated by the presence of 25-hydroxycholesterol; 2) immunoblotting of P450scc indicated no stimulatory effect of estradiol on the amount of enzyme; and 3) levels of P450scc mRNA were not increased by estradiol. Whereas estradiol had no stimulatory effect on P450scc it caused a mark (3-fold) increase in the mitochondrial content of SCP2. Thus, the increase in luteal progestagen synthesis stimulated by estradiol appears to be associated with an increase in mitochondrial SCP2 and is independent of luteal P450 content or message.