Vasoactive intestinal peptide (VIP) and PRL have been reported to be colocalized in rat lactotropes. To determine whether induced hypothyroidism, known to reduce pituitary PRL concentration, also reduces pituitary concentration of VIP, rats were treated with antithyroid drugs for 3 weeks. Pituitary PRL concentration in male rats (micrograms/mg protein) was markedly reduced by this treatment (9.4 +/- 1.0 vs. 2.3 +/- 0.4 when extracted at pH 1.1, 17.9 +/- 3.0 vs. 3.4 + 0.4 when extracted at pH 7.4, 21.8 +/- 3.3 vs. 6.7 + 1.3 when extracted at pH 10.0). Contrary to expectation, pituitary VIP concentration was markedly increased in hypothyroidism; in males from 169.5 +/- 20.3 to 834.0 +/- 82.2 pg/mg protein, and in females (whose pituitary PRL had been similarly reduced) from 103.1/I +/- 34.1 to 771.6 +/- 100.9 pg/mg protein. Serum PRL was significantly reduced in hypothyroid males (7.4 +/- 1.6 vs. 28.9 +/- 12.2 ng/ml) whereas in females, serum PRL was not significantly altered (41.4 +/- 11.6 vs. 38.8 +/- 14.3 ng/ml). The effect of hypothyroidism was reversed by administration of T4 in physiological doses. The authenticity of pituitary immunoreactive VIP was further established by demonstrating chromatographic patterns by Sephadex G-50 gel exclusion and reverse phase HPLC separations identical to synthetic VIP. Immunohistochemically reactive VIP cells could not be demonstrated in normal pituitaries, but the marked increase in VIP in hypothyroid animals made it possible to visualize a population of VIP immunoreactive stellate cells which appear to be distinct from hypothyroid lactotropes and thyrotropes.