Ganglia expressing the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) innervate vasoactive intestinal peptide (VIP) containing neurons suggesting a role of PACAP in regulating VIP expression. Human NB-1 neuroblastoma cells were applied to study PACAP regulated VIP gene expression aiming to identify the receptor and the signaling proteins involved. The PACAP receptor subtype PAC1 induced VIP gene expression as (i) PACAP and the PAC1 receptor agonist maxadilan were equally efficient and approximately 200-fold more potent than VIP, and (ii) PACAP6-38 and PG99-465, antagonists of PAC1 and VPAC2 receptors, respectively, abolished and did not affect the PACAP-induced VIP mRNA expression, respectively. A pivotal role of PKA was implicated in addition to partial involvement of PKC and ERK1/2 in PACAP-induced VIP gene expression as H-89, Bisindolylmaleimide I (BIS), Gö6976 and U0126 attenuated the VIP mRNA expression by 93%, 58%, 58% and 40%, respectively. PACAP modulated the phosphorylation of ERK1/2 (pERK1/2) and CREB/ATF-1 (pCREB/ATF-1) concomitant with a translocation of PKA to the nucleus. Inhibition of conventional PKC isoforms and MEK1/2 completely abolished pERK1/2 without affecting PACAP induced pCREB/ATF-1. In contrast, inhibiting PKA attenuated PACAP induced pCREB/ATF-1. PACAP also enhanced the FOS gene expression and individual presence of H-89, BIS, Gö6976 and U0126 partially attenuated the PACAP induced FOS mRNA expression. Combining the kinase inhibitors completely suppressed the PACAP induced FOS mRNA expression. Immunoblotting confirmed expression of FOS protein upon addition of PACAP, which was diminished by impairment of PKC, ERK1/2 and PKA activities. The resemblance of the signaling pathways involving concomitant activities of PKC, ERK1/2 and PKA in PACAP regulation of the FOS and VIP gene expressions suggest for the first time a role of FOS in PACAP-induced VIP gene expression in human NB-1 neuroblastoma cells.