Free radicals may produce cytotoxicity to pancreatic islets under pathophysiological conditions. The aim of our in vitro investigations was to compare functional and morphological changes in pancreatic beta-cells induced by reactive oxygen species (ROS) generated by alloxan or xanthine oxidase/hypoxanthine (XO/HX), respectively. We demonstrate that short-term exposure to alloxan or to XO/HX leads to a temporarily elevated insulin release from isolated pancreatic islets. On application of alloxan, this effect is caused by beta-cell necrosis and can be prevented by administration of melatonin, while in contrast, XO/HX did not lead to long-term morphological changes in the majority of the cells. Among the cells destroyed by alloxan, only necrosis could be detected, while in contrast, some apoptotic cells were identified by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction and electron microscopic examinations of cells treated with XO/HX. Melatonin was able to prevent the changes caused by alloxan, but failed to influence the alterations caused by XO/HX. Using electron spin resonance and lipid peroxidation assay, respectively, it was confirmed that melatonin effectively detoxifies hydroxyl radicals. Therefore, we believe that hydroxyl radicals are the toxic principle of alloxan, but not of XO/HX toxicity.