The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are widely expressed in the nervous system and various other tissues. PACAP exerts strong anti-apoptotic effects in neuronal cell lines and, according to recent data, also in non-neuronal cells. The peptide is present in the cardiovascular system and has various distinct effects. We have demonstrated earlier that PACAP has protective effects against in vitro ischemia/reperfusion-induced apoptosis in cardiomyocytes. Preconditioning with brief intermittent periods of ischemia is known to provide protection against ischemic injury. The aim of the present study was to investigate whether PACAP could enhance the protective effect of preconditioning against in vitro ischemic injury. Cultured cardiomyocytes were exposed to brief preconditioning ischemia followed by 2 h ischemia and 4 h reperfusion. Both PACAP treatment and preconditioning alone significantly increased cell viability and decreased the ratio of cell death. Pretreatment with PACAP was found to further reduce the level of cleaved caspase-8 but it did not lead to additional survival rate when compared to cells treated with PACAP or preconditioning alone. These results show that although both PACAP and preconditioning have a protective effect against ischemia/reperfusion-induced cardiomyocyte apoptosis, their effects are not additive.