The immunocytological method was used to investigate whether vasoactive intestinal peptide (VIP) is present in the pituitary gland and to localize the peptide at the cellular and subcellular levels. Pituitaries of Wistar male and female rats (Iffa Credo) were fixed in glutaraldehyde 2.5% and postosmicated and frozen in liquid nitrogen. Ultrathin slices, obtained by cryo-ultramicrotomy were incubated with the antiserum. The antigen-antibody reaction was detected by peroxidase-antiperoxidase complexes revealed by 4-chloro-1-naphtol. The prolactin (PRL)-secreting cells were identified by using an anti-oPRL antiserum. The PRL immunoreactivity was localized in secretory granules of irregular shapes. An anti-VIP serum was used which neither cross-reacted with the several fragments of VIP molecule nor with peptides from gut or hypothalamus. The VIP immunoreactivity obtained with this antiserum, was observed in PRL cells only but never in so-matotropic, gonadotropic, corticotropic and thyrotropic cells. The immunoreactivity was localized in the cytoplasmic matrix between and around the secretory granules but not in the organelles, and in the nucleus distributed all over the euchromatin near to the heterochromatin regions. No reaction was observed by using either nonimmune serum or anti-VIP antiserum incubated with VIP. No modification of VIP immunoreactivity was observed by using anti-VIP antiserum incubated with somatostatin, gonado- or thyroliberin. These data (1) provide immunocytological evidence for presence of VIP in pituitary gland; (2) indicate the presence of this peptide in one particular pituitary cell type, and (3) support the hypothesis that VIP could have a direct effect on the control of PRL secretion.