The 38 amino acid neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP-38) induced differentiation of human neuroblastoma SH-SY5Y cells. Four-day treatment with PACAP-38 caused a concentration-dependent increase in neurite-bearing cells (5.6 ± 1.0-fold of basal control at 100 nM; n=3) and a marked increase in the expression of the neuronal differentiation marker proteins, Bcl-2, GAP-43 and ChAT, as assessed by Western blotting (n=3). These effects were blocked by the PAC₁ receptor antagonist, PACAP6-38. The PAC₁ receptor couples to the robust activation of adenylyl cyclase [Lutz et al., 2006], and the effects of PACAP-38 on SH-SY5Y cells could be mimicked by forskolin treatment, suggesting the involvement of a cAMP-mediated pathway. PACAP-38-induced differentiation was completely prevented by the adenylyl cyclase inhibitor, DDA, but not by the PKA inhibitor H89, or siRNA-mediated knock-down of the PKA catalytic subunit. Both PACAP-38 and forskolin were observed to stimulate the activation of ERK, p38 MAP kinase and JNK. PACAP-38-induced neuritogenesis was completely prevented by the MEK-1 inhibitor PD98059, and partially prevented by the p38 MAP kinase inhibitor SB203580. Activation of the guanine nucleotide exchange factor, EPAC (exchange protein directly activated by cAMP), by the specific activator 8-CPT-Me-cAMP, also partially mimicked the effects of PACAP-38, and led to the phosphorylation of ERK but not p38 MAP kinase. These data therefore indicate that PACAP-38, acting at the PAC₁ receptor, is a neurotrophic factor and that the mechanism involved is cAMP-dependent but PKA-independent, and involves EPAC-dependent activation of ERK as well as activation of p38 MAP kinase signalling pathways.