The effects on ovulation at the next estrus after unilaterally implanting pilocarpine in the preoptic-anterior hypothalamic area (POA-AHA) of rats on each day of the estrous cycle were analyzed. Implantation on the left side of POA-AHA on the day of estrus blocked ovulation in all animals, whereas implantation on the right side did not (0/5 vs. 4/4, P < 0.05). Implantation on diestrus 1 or 2 on either side of the POA-AHA blocked ovulation. Implantation on the right side of the POA-AHA at the day of proestrus blocked ovulation (1/6 animals ovulated), while 10/12 with pilocarpine on the left side ovulated (P < 0.05). The administration of 3.7 micrograms of GnRH at 13:00 h on the expected day of proestrus induced ovulation in 36/42 treated animals. In rats with a pilocarpine implant, the injection of estradiol benzoate on diestrus 2 restored ovulation only in those animals with the pilocarpine implant placed in the left side of the POA-AHA, performed on the day of estrus. The results support the previous statements that in the adult rat POA-AHA, the cholinergic mechanism regulating preovulatory GnRH release, is lateralized. In addition, at the beginning of the estrous phase, the POA-AHA-cholinergic system needs to remain undisturbed for normal ovulation to take place at the next estrus.