Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are hypothalamic factors believed to play a role in the regulation of anterior pituitary cell function. However, little is known about the expression of PACAP/VIP receptor (PVR) subtypes in such cells. Three PVR subtypes have recently been cloned: the PACAP-selective PVR1, and PVR2 and PVR3, which exhibit similar affinities for PACAP and VIP. In the present study we used the reverse transcription-polymerase chain reaction with PVR-specific primers to identify the PVR messenger RNAs (mRNAs) expressed in the somatotroph-like GH4C1 and the gonadotroph-like alpha T3-1 cell lines. In parallel, the effects of PACAP and VIP on intracellular signaling were studied. GH4C1 cells were found to express mRNA only for the PVR3, and neither PVR1 nor PVR2 mRNA was found. PACAP and VIP stimulated Ca2+ influx responses in individual GH4C1 cells and were equipotent in stimulating cAMP production (EC50, 15 nM) in GH4C1 cell populations, but failed to stimulate inositol phospholipid (PI) turnover, results consistent with the expression of a PVR3. In contrast, alpha T3-1 cells expressed mRNA for PVR1 and PVR3, but not PVR2. The predominant splice variant forms of PVR1 observed were PVR1s and PVR1hop, although the other forms (PVR1hiphop and PVR1hip) were also seen at much lower levels. PACAP stimulated a Ca2+ store-dependent Ca2+ spike and a sustained Ca2+ influx in individual alpha T3-1 cells, whereas VIP only stimulated Ca2+ influx. PACAP (EC50, 3 nM) was approximately 1000-fold more potent than VIP (EC50, approximately 3 microM) in stimulating cAMP production. PACAP also stimulated PI turnover (EC50, approximately 20 nM), whereas VIP stimulated PI turnover only at very high (10 microM) concentrations. These results are indicative of the expression of a PVR1. Rat anterior pituitary tissue expressed mRNAs for PVR1, PVR3, and low levels of PVR2. The coexpression of different PVRs in the same cell type and the differential expression of PVRs in different cell types would allow for a complex regulation of anterior pituitary gland function by PACAP and VIP.