Vasoactive intestinal peptide (VIP), a prolactin (PRL)-releasing factor, has been shown to be synthesized within the anterior pituitary. To test the hypothesis that estrogens increase PRL secretion, at least in part, by stimulating VIP secretion, the concentrations of VIP, peptide histidine isoleucine (PHI) and prepro VIP mRNA were measured in the anterior pituitaries of oophorectomized rats treated with 17 beta-estradiol benzoate 25 micrograms/kg/day s.c. for 5 days. For comparison, changes in the hypothalamus were also measured. Estrogen treatment resulted in a marked increase in pituitary VIP content without detectable changes in PHI content, suggesting that estrogen may regulate differentially the enzymes involved in the posttranslational processing of the VIP prohormone. A VIP mRNA-transcript of about 1.7 kilobases was detected in all tissues studied, being most abundant in the cortex, less abundant in the hypothalamus and barely detectable in the untreated pituitary. Estrogen treatment resulted in an increase in VIP gene expression in the pituitary but not in the hypothalamus or cerebral cortex. This marked increase in prepro VIP mRNA rendered possible the demonstration in the estrogen-treated pituitary of a second VIP transcript of about 1.0 kilobase which was present in only very low quantities in the cortex and hypothalamus. We conclude that estrogen regulates the gene expression of VIP in the anterior pituitary. Changes in VIP secretion may contribute to the stimulatory effect of estrogen on PRL secretion.