The regulation of the two isoforms of prostaglandin endoperoxide synthase (PGS-1 and PGS-2) and prostaglandin synthesis by luteinizing hormone (LH)/3-isobutyl-1-methylxanthine (IBMX) and progesterone was examined in granulosa cells and residual ovarian tissue of rat ovaries perfused in vitro. The endogenous progesterone synthesis was blocked by an inhibitor of 3 beta-dehydroxysteroid-dehydrogenase, compound A (CA), previously shown to reversibly inhibit ovulation in the in vitro perfused rat ovary. Preovulatory ovaries were perfused for 7 h, and soluble extracts from granulosa cells and residual ovarian tissue were obtained for immunoblotting and determination of the tissue contents of PGS-1/PGS-2. The tissue concentrations of prostaglandins (PGE2, PGF2 alpha and 6-keto-PGF1 alpha) were measured. The ovaries were perfused with medium alone (control) or medium containing LH (0.1 microgram/ml) and IBMX (0.2 mM), LH+IBMX+CA (10 micrograms/ml) or LH+IBMX+CA+progesterone (10 micrograms/ml). PGE2, PGF2 alpha and 6-keto-PGF1 alpha tissue concentrations were increased by LH+IBMX, with highest values detected for PGE2. The addition of CA alone or CA in combination with exogenous progesterone, did not change the values of prostaglandins increased by LH+IBMX. The content of PGS-1 was only marginally changed in both granulosa cells and residual ovarian tissue in the different treatment groups, compared to the control group. In contrast, PGS-2 was markedly increased by LH+IBMX, especially in the granulosa cells. The addition of CA, in combination with LH+IBMX, resulted in a small decrease of PGS-2, and progesterone further decreased its content. In the residual ovarian tissue, only minor changes of PGS-2 were detected. These results demonstrate that LH and progesterone selectively regulate the expression of PGS-2 in rat granulosa cells, whereas the hormonal regulation of PGS-1 is less pronounced. Progesterone inhibits PGS-2 in granulosa cells but has negligible effects on the total ovarian synthesis of prostaglandins during the ovulatory period.