Calcitonin gene-related peptide (CGRP) has been found in nerves that innervate the rat ovary. In this study we used indirect immunohistochemical methods to investigate the normal distribution of CGRP-immunoreactive fibers in the prepubertal rat ovary and to determine the route by which these fibers reach the gland. Additional experiments were performed to examine the possible colocalization of CGRP with immunoreactivities of substance P-, neuropeptide Y-, and tyrosine hydroxylase. Potential effects of CGRP on estradiol and progesterone secretion were explored with cultured granulosa cells and short-term incubation of whole ovaries in vitro. In ovaries with intact nerves, CGRP-labeled fibers were observed in dense plexuses surrounding the ovarian vasculature. Additional fibers were found occasionally in the interstitial tissue and in the vicinity of ovarian follicles. Surgical transection of the plexus nerve completely eliminated CGRP immunoreactivity. Section of the superior ovarian nerve or the abdominal vagal trunks had no discernible effect on CGRP labeling. Additional studies revealed coexistence of CGRP and substance P in several axons. Neither tyrosine hydroxylase nor neuropeptide Y-immunoreactivity was co-localized in CGRP-containing fibers. CGRP (10(-10) - 10(-6) M) had no effect on the basal or follicle-stimulating hormone-stimulated release of estradiol or progesterone from ovaries of pregnant mare's serum gonadotropin-treated rats or from cultured granulosa cells from hypophysectomized, diethylstilbestrol-treated rats. We conclude that CGRP-containing nerves enter the ovary via the plexus nerve and are probably involved in the regulation of vasomotor function.