2-Hydroxyestradiol (2-OH-E2) stimulates progestin secretion by granulosa cells, but the intracellular locus of the stimulatory effect has not been clarified. The objectives of the present studies were to 1) determine the role of de novo sterol synthesis in the effect of 2-OH-E2 on progestin biosynthesis, and 2) examine the effects of 2-OH-E2 on cholesterol side-chain cleavage (SCC) activity and the level of messenger RNA (mRNA) for P450scc. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase with lovastatin (5 micrograms/ml) or mevinolin (5 micrograms/ml) reduced FSH- and 2-OH-E2-stimulated (but not E2-stimulated) progesterone production. Mevalonate (20 mM) enhanced basal progesterone production and reversed the inhibitory effect of lovastatin but did not affect progesterone biosynthesis in the presence of 2-OH-E2. As an index of the activity of cholesterol SCC enzyme, granulosa cells were exposed to 25-hydroxycholesterol (10 micrograms/ml) for 24 h and progesterone secretion monitored. Conversion of 25-hydroxycholesterol into progesterone was stimulated 2- to 3-fold by maximally effective concentrations of 2-OH-E2, E2, and FSH. 2-OH-E2 and/or E2 further enhanced 25-hydroxycholesterol conversion in the presence of FSH, LH, and epinephrine. Aminoglutethimide, an inhibitor of SCC, reduced 2-OH-E2- and 2-OH-E2 plus FSH-stimulated progesterone production by 97% and 95%, respectively. 2-OH-E2 also increased basal (by 2 to 3-fold) and FSH-stimulated (to 3.5-fold of FSH-treated controls) levels of mRNA for cytochrome P450scc. Collectively, our studies support the hypothesis that 2-OH-E2-enhanced progesterone biosynthesis by porcine granulosa cells is dependent on de novo cholesterol synthesis and is associated with increased levels of the mRNA encoding cytochrome P-450scc, which leads to increases in basal and gonadotropin-induced SCC activity.