Melittin, a cationic antimicrobial peptide isolated from the venom of Apis mellifera, has shown potential as a permeability enhancer, transiently increasing intestinal permeability and enhancing the absorption of paracellular markers. Although it is cytotoxic to eukaryotic cells, its cytotoxicity is significantly lower in polarised epithelia compared to non-polarised cells. The aim of this study was to explore the mechanism of melittin cytotoxicity in gastrointestinal cells and to determine whether cytotoxicity was mediated by a necrotic or an apoptotic pathway. The role of cholesterol in melittin cytotoxicity was also examined. Using four distinct assays for apoptosis, phosphatidylserine translocation, caspase activation, DNA ladder formation and cell cycle analysis, no evidence of apoptotic pathway for cell death was observed with any of these approaches. It can therefore be concluded that cytotoxicity was likely to be mediated by necrosis in gastrointestinal epithelial cells. However, at low concentrations of melittin (<1 microM), BRDU uptake was enhanced, demonstrating proliferative effects of melittin at sub-lethal concentrations. Furthermore, melittin cytotoxicity was further enhanced by depletion of cholesterol, using methyl-beta-cyclodextrin, indicating that cholesterol depleting agents could be contradictory to its potential as an enhancer. Overall, although melittin appears to stimulate necrosis, with careful dosage selection the peptide could be considered for the oral delivery of poorly bioavailable drugs.