To clarify a regulatory role of prostaglandins in collagenolytic enzyme activities of the follicular wall at ovulation, we studied whether the ovulation-blocking effect of indomethacin (IM) on hCG-stimulated rabbits could be reversed by an intravenous infusion of PGF2 alpha. The activities of BANA hydrolase and DNP peptidase were also measured by using synthetic substrates N-benzoyl-DL-arginine-2-naphthylamide HCl (BANA) and DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH (DNP peptide), respectively, every hour during the specific preovulatory period. The ovulation-blocking effect of IM (4mg/kg) was reversed by an intravenous infusion of PGF2 alpha (1.5 mg/kg) starting at 3 hr after the hCG injection. In the control rabbits treated with hCG(100 iu) alone, these enzymatic activities were increased toward ovulation with the peak level 7-9 hr after a hCG injection and decreased significantly at 10 hr. The preovulatory increase in these enzymatic activities disappeared following the ovulation-blocking doses of indomethacin. By the addition of the PGF2 alpha infusion, the IM-induced changes in BANA hydrolase and DNP peptidase activities were returned close to the control. It is suggested that the preovulatory increases in BANA hydrolase and DNP peptidase are mandatory for ovulation and PGF2 alpha facilitates ovulation through its regulatory action on these enzymes.