To determine whether brain monoaminergic neurons are involved in the release of gonadotropins responsible for estrogen increases before proestrus, various inhibitors and precursors of monoamine biosynthesis were administered subcutaneously or intracranially to the 3rd ventricle at 10.00 or 20.00 on the day before proestrus, the 2nd day of diestrus (DII) in 4-day cycling rats. The inhibitors used were α-methyl-p-tyrosine (α-MPT) and bis-(4-methyl-1-homopiperazinyl-thiocarbonyl)-disulfide (FLA-63). The effects of these drugs on changes in vaginal cytology, ovulation, uterine weight, weight of uterine intraluminal fluid, and on serum concentrations of LH and FSH were evaluated in selected experiments. (1) Administration of α-MPT (150 mg/kg s.c), an inhibitor of tyrosine hydroxylase, at 10.00 on DII reduced weights of uterus and intraluminal fluid on the day of expected proestrus (P), prevented vaginal cornification on estrus (E), blocked ovulation in all 10 rats, and induced prolonged diestrus. (2) Administration of FLA-63 (10 mg/kg s.c), an inhibitor of dopamine-β-hydroxylase, at 10.00 on DII reduced weights of uterus and intraluminal fluid on P, blocked ovulation for a few days but did not prevent vaginal cornification at the expected time of E. (3) Administration of α-MPT (200 mg/kg) or FLA-63 (15 mg/kg) at 20.00 on DII blocked ovulation in all of 8 and 7 rats, respectively, but these treatments did not block vaginal cornification at the expected time in any animal. (4) Administration of L-DOPA (100 mg/kg) or dihydroxy-phenylserine (DOPS, 200 mg/kg) with α-MPT (200 mg/kg) at 20.00 on DII reversed the blocking effect of α-MPT on ovulation in 3 out of 6 and 3 out of 5 rats, respectively. (5) Direct application of crystalline α-MPT or FLA-63 (about 3–5 µg) to the 3rd ventricle at 20.00 on DII also blocked ovulation in all of 7 and 5 rats, respectively. (6) Both systemic and intraventricular