The aim of the present study was to investigate whether exposure of porcine ovarian follicular cells to clinically relevant concentrations of valproate affected steroid production in vitro and to which extent these effects were reversed by removal of the drug from the culture medium. Small and medium follicles were obtained from ovaries collected, respectively, at days 8-10 and 14-16 of the estrous cycle. Theca and granulosa cells were collected from follicles and co-cultured in one well. To show whether the effect of valproate was reversible, cells were cultured for 24, 48, or 72 h with valproate 100 or 250 microg/ml. The medium was then changed to fresh medium without drugs for an additional 24, 48, or 72 h. Valproate added to the culture medium caused a significant reduction of estradiol secretion with concomitant increase in both testosterone and progesterone secretion by small follicles. In medium-sized follicles, 100 microg/ml valproate was without effect on estradiol secretion while 250 microg/ml caused a small, but statistically significant decrease. The effects of valproate on steroid secretion patterns were irreversibly independent of concentration, exposure time, and time of restoration after drug exposure up to 72 h in both small and medium follicles. In conclusion, the present study demonstrated that even short-term valproate treatment disrupted follicular steroidogenesis in isolated ovarian follicular cells resulting in increased testosterone and progesterone and decreased estradiol secretion. It was not possible to reverse the steroidogenic effects of valproate by removing the drug from the cell cultures.