It is well established that oestrogens can stimulate prolactin (PRL) secretion as well as the expression of the vasoactive intestinal peptide (VIP) gene whose product is also a potent PRL secretagogue. Previous evidence has supported both an autocrine and a paracrine role for pituitary VIP in PRL release in vitro; however, the cellular origin of VIP in pituitary tissue still remains poorly defined. In these studies, we have demonstrated by in situ hybridisation that VIP RNA is detected in the anterior pituitaries of chronically hyperoestrogenised rats, but not in those of untreated animals. Using a double-probe labelling procedure, VIP RNA has been shown to be present in a subpopulation of PRL-producing cells, while colocalisation of VIP and GH RNA was not observed. VIP gene expression in the rat anterior pituitary gland was characterised by the presence of two alternatively polyadenylated transcripts, 1·7 kb and 1·0 kb in size. We have generated a probe specific for the 1·7 kb transcript and double-labelling studies also showed definitive colocalisation with PRL mRNA. Our results demonstrating the presence of VIP RNA in PRL-producing cells thus suggest that VIP may play an autocrine role in PRL hypersecretion under conditions of oestrogen-induced hyperplasia.