Vasoactive intestinal peptide (VIP) and peptide (P) with N-terminal histidine and C-terminal isoleucine (PHI) stimulated prolactin (PRL) secretion from GH4C1 cells equipotent with ED50 values of 30-50 nM. In a parafusion system optimized to give high time resolution both VIP and PHI increased PRL secretion with a delay of about 60 s and subsequent to the activation of the adenylate cyclase. Thyroliberin (TRH) increased PRL secretion within 4 s. The dose-response curves for VIP- and PHI-stimulated cAMP accumulation were superimposable on those for PRL secretion. At submaximal concentrations the effects of VIP and PHI on both cAMP accumulation and PRL secretion were additive, whereas the effects were not additive at concentrations giving maximal effects. VIP and PHI increased [Ca2+]i measured by quin-2 in a different way than TRH, without inducing changes in the electrophysiological membrane properties of the GH4C1 cells. We conclude that both VIP and PHI stimulate PRL secretion via a cAMP-dependent process involving an increase in [Ca2+]i.