Numerous investigations have demonstrated that T cells are involved in destruction of β cells in the NOD mouse, a widely studied model of type I diabetes. In this report we describe a series of islet‐specific T cell lines established from islet‐infiltrating lymphocytes obtained from individual pre‐diabetic NOD mice as well as a large panel of clones derived from these lines. Proliferation assays indicated that these nominally islet‐specific lines responded vigorously to porcine insulin. Furthermore, of 40 islet‐specific clones derived from lines established from 12‐week‐old mice, 22 (55%) responded to insulin. A similar analysis of islet‐specific clones established from 7‐week‐old mice indicated that 2 of 14 (14%) were insulin specific. These findings demonstrate that insulin‐specific T cells can comprise a major portion of the spontaneously arising T cell response to islets in NOD mice.