Islet-reactive T-cell clones from NOD mice provide an important approach to the investigation of antigens with relevance to type I diabetes. To identify a source of β-cell antigen suitable for biochemical studies, we have used two islet-specific, diabetogenic T-cell clones to test β-tumor cells. β-tumor cell lines, maintained in continuous culture, were found to lose antigenicity rapidly. However, cells harvested directly from β-tumors arising spontaneously in the transgenic NOD/Lt-Tg(RIPTag)1Lt mouse proved to be a potent source of β-cell antigen for the T-cell clones. Subcellular fractionation of β-tumor cells showed that the T-cell antigen was highly enriched in the β-granule fraction and that this activity was associated with the granule membrane.