T-cells have been shown to cause insulitis and ultimately be responsible for the destruction of β-cells in animal models of insulin-dependent diabetes mellitus (IDDM). In one murine model, insulitis and hyperglycemia occur after administration of five low doses of streptozotocin (STZ) (multidose STZ-induced diabetes mellitus [MDSDM]). Insulitis can first be identified in the islets after the final (fifth) daily dose of STZ is given. We have studied the T-cells that infiltrate the islets of Langerhans during the early stages of diabetes by preparing Southern blots of T-cell receptor (TCR) β-chain genes amplified by polymerase chain reaction (PCR) from islets from C57BL/KsJ mice given multiple doses of STZ. The relative abundance of TCR gene products in islets was compared with spleen cells stimulated with anti-CD3 monoclonal antibody (mAb). We found that after the fourth dose of STZ, there was a striking increase in the amount of Vβ8.2 TCR gene product (37 ± 4% of total PCR signal) compared with T-cells in the spleen (9 ± 2%, P < 0.01), which increased further 2 days after the final dose of STZ (47 ± 5%, P < 0.001). We studied the heterogeneity of the size of the Vβ8.2 TCR CDR3 region and found primarily products with only two lengths compared with a heterogeneous population in the spleen. Treatment with anti-Vβ8 mAb, but not anti-Vβ9 and anti-Vβ13 mAbs, prevented development of hyperglycemia (P < 0.0001) and insulitis (P < 0.0005) after STZ administration. We conclude that there is limited heterogeneity of the T-cell response that causes diabetes in MDSDM. A limited number of Vβ8.2+ cells are preferentially expanded in the islets in the early stages. Our results suggest that in this model of IDDM, a limited number of T-cells cause the disease.