The objective of the study was to evaluate the effect of valproic acid (VPA) on ovarian androgen biosynthesis in primary cultures of theca-interstitial (T-I) cells isolated from rat ovaries. Ovarian T-I cells were cultured with VPA in the presence or absence of hCG. VPA did not increase basal or hCG-stimulated androgen synthesis when added to primary cultures of T-I cells. However, the addition of VPA caused a marked concentration-dependent inhibitory effect on hCG-stimulated androstendione synthesis. Treatment of T-I cells with 8-Bromo-cAMP resulted in a marked increase in the production of androstenedione, and VPA inhibited this stimulatory effect, suggesting that the mechanism of VPA's inhibitory effect on androstenedione production occurs at a step after second messenger activation. Treatment of T-I cells with hCG resulted in a significant increase in the mRNA expression of steroidogenic enzymes CYP17A1 and 17β-hydroxysteroid dehydrogenase. Addition of VPA sharply blunted the stimulatory effect of hCG, reducing the mRNA expression of the steroidogenic enzymes to basal levels. In conclusion, VPA exerts an inhibitory effect on hCG-stimulated androgen synthesis in rat T-I cells.