T cells are known to play an important role in beta cell destruction in the nonobese diabetic (NOD) mouse model of Type I diabetes and islet-specific T cell clones have been demonstrated to be capable of adoptive transfer of diabetes. One important issue involves the identity of beta cell antigens that are recognized by nominally islet cell-specific T cell clones. We have previously reported that insulin-specific T cells are a predominant component of islet-specific T cells isolated from islet infiltrates of pre-diabetic NOD mice. In this report we examine six independently derived insulin-specific T cell clones established from islet infiltrates of pre-diabetic NOD mice in detail. All six clones were found to be specific to a region of the insulin molecule defined by a synthetic peptide encompassing residues 9-23 of the B chain. Despite this restricted specificity, each member of this panel exhibited a distinct receptor specificity defined either by V beta usage or antigen fine specificity. Five clones produced interferon (IFN)-gamma but not interleukin (IL)-4, placing them in the T helper type 1 (TH1)-like category whereas one clone produced both IL-4 and IFN-gamma, a characteristic of TH0 cells. All six clones were capable of either acceleration of diabetes in young NOD mice or adoptive transfer to NODscid mice. Taken together, these results suggest that spontaneously arising insulin-specific T cells participate in beta cell destruction during development of diabetes in NOD mice.