Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide expression of which has been found in various tumors of the brain and peripheral organs. Despite numerous studies, the exact role the peptide plays in the development and progression of tumors is not fully understood. In the present study, we investigated the effect of PACAP on human retinoblastoma Y79 cell viability. We found that both PACAP38 and PACAP6-38, a selective PAC1 receptor antagonist, did not affect Y79 cell viability at nanomolar concentrations, but when used at 1-5 μM potently reduced cell survival in a dose-dependent manner. PACAP27 and maxadilan, a high affinity agonist of PAC1 receptors, had negligible effects. Two membrane-penetrating analogs of PACAP38 inactive at PAC1/VPAC receptors, [Disc(6)]PACAP38 and FITC-Ahx-PACAP11-38, also decreased viability of Y79 cells, albeit with lower potency than PACAP38. The cytotoxic effect of PACAP38 was augmented by p38, MEK1/2, and JNK inhibitors, indicating that high concentrations of the peptide might decrease the activity of these kinases, leading to cell death. It is suggested that the cytotoxic activity of PACAP38 and PACAP6-38 against human retinoblastoma Y79 cell line may result from their interaction with target sites other than PAC1 and VPAC receptors, but this is yet unknown.