Localization of calcitonin gene-related peptide (CGRP) expression in the rat anterior pituitary and its changes during ontogeny and after gonadal steroid manipulations were studied by immunocytochemistry, RIA, and in situ hybridization. Colocalization studies and the combined use of immunocytochemistry and in situ hybridization revealed that CGRP immunoreactivity is localized mainly in gonadotropes and alpha- and beta-CGRP messenger RNAs were detected in CGRP-immunoreactive cells. Immunoreactivity for CGRP also was detected in nerve fibers and colocalized with substance P immunoreactivity. Cells immunoreactive to CGRP antiserum were first detected in fetal rats at gestational day 18, and the incidence considerably increased between postnatal days 5 and 14. CGRP immunoreactivity was low in control adults of both sexes and in pregnant and ovariectomized females but increased in lactating, estrogen-supplemented ovariectomized and high-dose estrogen-treated females, and in high-dose estrogen-treated and castrated males. Testosterone supplement suppressed the effect of castration on CGRP immunoreactivity in males. Quantities of extractable immunoreactive CGRP under conditions of estrogen manipulation corresponded well to the immunocytochemical findings (females: controls, 96.4 +/- 13.1 fmol/gland; ovariectomized, 107.6 +/- 19.2; high-dose estrogen-treated, 212 +/- 23.0; estrogen-supplemented ovariectomized, 680 +/- 42.1). The present study suggests that pituitary CGRP is synthesized and stored in gonadotropes, is modulated by gonadal steroids, and may have a functional link with gonadotropins.