Streptozocin (STZ) and alloxan (ALX) exhibit the most potent diabetogenicity and are used for induction of experimental diabetes mellitus. An understanding of the mechanisms of action of the typical diabetogenic agents is important for elucidating the causes of diabetes. Okamoto proposed a model in which DNA fragmentation plays an important role in the development of diabetes. DNA fragmentation supposedly results from the accumulation of superoxide or hydroxyl radicals. However, direct evidence for this accumulation is lacking. With isolated rat pancreatic islets in vitro, we demonstrated that STZ and ALX stimulated H₂O₂ generation and caused DNA fragmentation. Addition of STZ or ALX resulted in an increase in H₂O₂ generation. On DNA analysis, when incubated without STZ or ALX, DNA sedimented as a single peak; when incubated with STZ or ALX, DNA sedimented slower as a broad peak and was fragmented. Graded doses of STZ and ALX stimulated H₂O₂ generation and induced DNA fragmentation; their effects on H₂O₂ generation and DNA fragmentation were evident at a concentration of 0.1 mM and were maximal at 1 mM. Administration of STX or ALX to rats in vivo stimulated H₂O₂ generation and caused DNA fragmentation in pancreatic islets. H₂O₂ itself also induced DNA fragmentation. These findings may support Okamoto's proposal that STZ and ALX induce diabetes through the following biochemical events: STZ and ALX→H₂O₂ generation→DNA fragmentation→β-cell destruction. This study may constitute the first demonstration of STZ- and ALX-stimulated H₂O₂ generation, which probably acts as a mediator of STZ- and ALX-induced DNA fragmentation.