The present studies were undertaken to determine whether nitric oxide (NO) is involved in the regulation of ovarian progesterone and oestradiol secretion in rats. Immature female Sprague–Dawley rats at 27 days of age were injected s.c. with 4 IU pregnant mare's serum gonadotrophin (PMSG) and were killed 72 h after the injection. The ovaries were collected, weighed and cultured in Dulbecco's modified Eagle's medium containing saline, NO donor, NO synthesis inhibitor or prostaglandin F2α (PGF2α). After 24 h culture, the medium concentrations of progesterone and oestradiol were measured by radioimmunoassay. Results showed that: (i) diethylenetriamine (DETA)/NO (1×10–6, 1×10–5, 1×10–4 M), an NO donor, caused a dose-dependent increase in progesterone synthesis (355 ± 43, 443 ± 46, 647 ± 55 ng/g ovary respectively, P < 0.01) with a concomitant decrease in ovarian oestradiol secretion (408.1 ± 50.7, 272.9 ± 28.2, 132.6 ± 34.6 pg/g ovary respectively, P < 0.01); (ii) neither progesterone nor oestradiol concentrations in the culture medium were altered by DETA without NO; (iii) NG-nitro-l-arginine methyl ester (1×10–4 M), an inhibitor of NO synthesis, did not significantly affect progesterone and oestradiol secretion by rat ovaries; (iv) PGF2α (1×10–6 M) caused a fall in progesterone and oestradiol synthesis; (v) co-incubation with DETA/NO, significantly reversed the PGF2α-induced decrease in progesterone concentrations from 184 ± 29 to 388 ± 60 ng/g (P < 0.01), but not that of oestradiol. It can be concluded that NO up-regulates progesterone secretion and down-regulates oestradiol secretion in rat ovaries, and NO can reverse PGF2α-induced inhibition in ovarian progesterone secretion.