In addition to the genetic framework, there are two other critical requirements for the development of tissue-specific autoimmune disease. First, autoreactive T cells need to escape thymic negative selection. Second, they need to find suitable conditions for autoantigen presentation and activation in the target tissue. We show here that these two conditions are fulfilled in diabetic mice of the nonobese diabetic (NOD) strain. A set of autoreactive CD4(+) T cells specific for an insulin peptide, with the noteworthy feature of not recognizing the insulin protein when processed by antigen-presenting cells (APCs), escaped thymic control, participated in diabetes and caused disease. Moreover, APCs in close contact with beta cells in the islets of Langerhans bore vesicles with the antigenic insulin peptides and activated peptide-specific T cells. Our findings may be relevant for other cases of endocrine autoimmunity.