The immature rat that has been induced to ovulate with pregnant mare serum (PMS) has proven to be a valuable model for the study of antiovulatory compounds. This paper describes an extension of this model in order to attempt to study the site of action of substances such as pentobarbital and a pineal compound, melatonin. A first experiment was designed to define a specific time for injecting pentobarbital in order to inhibit ovulation. In this study immature female rats were given injections with 25 IU PMS; pentobarbital was given at various times after PMS treatment. This study showed that sodium pentobarbital (35 mg/kg, i.p.) inhibits LH release and ovulation when rats have been anesthetized between 2 and 6 p.m. on day 2 after PMS treatment. In a second experiment ovulation was blocked with pentobarbital (35 mg/kg, i.p., beginning at 12 noon and at 2 p.m. on day 2 after PMS treatment) and completely restored to normal with the s.c. injection of 2 µg GnRH at 2 and 4 p.m. on day 2 after PMS treatment. In the third experiment, varying doses of GnRH were studied for their capacity to overcome the pentobarbital block. This study showed that 2 µg, 1 µg, 500 ng, and 250 ng of GnRH at 2 and 4 p.m. on day 2 after PMS treatment were equipotent in causing ovulation. In a fourth experiment ovulation was blocked with melatonin and this block was overcome with exogenous GnRH. In the last study exogenous GnRH was shown to restore ovulation after being blocked by both melatonin and pentobarbital. This evidence suggests that pentobarbital and melatonin inhibit ovulation by inhibiting the secretion of endogenous Gn