Gonadotropin-releasing hormone (GnRH) has been found to be expressed within the ovary and to modulate cell differentiation in ovarian cells. In the present study we have analyzed the influence of GnRH on DNA synthesis in rat granulosa cells. Cells were obtained from immature DES-treated rats and cultured in defined medium (DMEM:F12) containing combinations of FSH, estradiol, and transforming growth factor-beta (TGF-beta), both in the presence and absence of GnRH. A GnRH analog, Leuprolide (GnRHa), caused a dose-dependent inhibition of 3H-thymidine incorporation in cells cultured in the presence of FSH (20 ng/ml) and TGF beta (2.5 ng/ml), at concentrations as low as 5 x 10(-11) M. Similarly, a complete inhibition of hormonally stimulated DNA synthesis were observed with another analog (Buserelin, ED50 = 1.58 +/- 0.22 x 10(-10) M) and native GnRH (ED50 = 1.4 +/- 0.3 x 10(-6) M). A competitive antagonist of GnRH (Antide) was used to neutralize the GnRH agonist effects. Antide 10(-8) M could prevent the inhibition elicited by 10(-7) M of Leuprolide. These results suggest that GnRH may play a role in the regulation of rat granulosa cell proliferation during follicular development.