The neurotrophic peptide PACAP (pituitary adenylate cyclase-activating polypeptide) elevates cAMP in PC12 cells. Forskolin and dibutyryl cAMP mimic PACAP's neuritogenic and cell morphological effects, suggesting that they are driven by cAMP. Comparison of microarray expression profiles after exposure of PC12 cells to either forskolin, dibutyryl cAMP, or PACAP revealed a small group of cAMP-dependent target genes. Neuritogenesis induced by all three agents is protein kinase A (PKA)-independent [not blocked by N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89)] and extracellular signal-regulated kinase (ERK)-dependent [blocked by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio) butadiene (U0126)], and therefore cAMP-dependent target genes potentially mediating neuritogenesis were selected for further analysis based on the pharmacological profile of their induction by PACAP (i.e., mimicking that of neuritogenesis). Small interfering RNA (siRNA) targeting one of these genes, Egr1, blocked PACAP-induced neuritogenesis, and siRNA targeting another, Vil2, blocked a component of the cell size increase elicited by PACAP. Neither siRNA blocked PACAP's PKA-dependent antiproliferative effects. PACAP signaling to neuritogenesis was also impaired by dominant-negative Rap1 expression but was not affected by inhibition of protein kinase C (PKC), indicating a G-protein-coupled receptor-mediated differentiation pathway distinct from the one activated by receptor tyrosine kinase ligands such as nerve growth factor (NGF), that involves both Rap1 and PKC. We have thus identified a cAMP-dependent, PKA-independent pathway proceeding through ERK that functions to up-regulate the transcription of two genes, Egr1 and Vil2, required for PACAP-dependent neuritogenesis and increased cell size, respectively. Dominant-negative Rap1 expression impairs both PACAP-induced neuritogenesis and Egr1 activation by PACAP, suggesting that cAMP elevation and ERK activation by PACAP are linked through Rap1.