We have previously identified the neurotransmitter vasoactive intestinal peptide (VIP) in nerve fibers of the immature rat ovary and showed that it stimulates steroid release by a mechanism involving increased synthesis of the components of the cholesterol side-chain cleavage enzyme complex. The present experiments were undertaken to study the ontogeny of ovarian VIP levels and to determine if they change in relation to the initiation of puberty. VIP was already detected in 2-day-old ovaries; levels remained constant at approximately 4.5 pg/mg ovary until the end of juvenile development (day 30). Thereafter, and preceding the peripubertal activation of the ovary, VIP levels increased two-fold, decreased gradually towards the first proestrus, and returned to juvenile values after the first ovulation. Transection of the ovarian nerves eliminated radioimmunoassayable VIP levels in both intact and hypophysectomized rats, indicating that ovarian VIP derives mostly from the extrinsic innervation of the gland. Treatment of hypophysectomized immature female rats with human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), growth hormone (GH), prolactin (Prl), estradiol, or their combination, failed to reproduce the peripubertal increase in VIP. In contrast, unilateral direct anodal current lesions of the left preoptic-anterior hypothalamic area (POA-AHA) of hypophysectomized juvenile rats led to a significant increase in VIP in the ipsilateral ovary. Surprisingly, both bilateral lesions and lesions of the right POA-AHA also increased VIP levels in the left ovary suggesting the existence of a marked asymmetry in the hypothalamic control of ovarian VIP. Lesions of the ventromedial nucleus, dorsal AHA or small lesions of the AHA were ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)