Nonobese diabetic (NOD) mice spontaneously develop autoimmune T cell‐mediated insulin‐dependent diabetes mellitus (IDDM). This unit presents a protocol for maintaining NOD mice under conditions permissive to full expression of their autoimmune potential. Methods are also described for diagnosing IDDM on the basis of glycosuria and glycosemia as well as for the semiquantitation of insulitis, a valuable subphenotype diagnostic of prediabetes in these mice, including a procedure for aldehyde fuchsin staining to identify β granules in β islet cells for diagnostic purposes. An adoptive‐transfer method is also included in which leukocytes, purified T cells, or T cell infiltrates obtained from the insulitic pancreas tissue of NOD mice are injected into prediabetic NOD or diabetes‐resistant F1 mice, which then develop disease in an accelerated fashion. This protocol also includes alternative steps in which bone‐marrow cells from NOD mice are transferred to syngeneic, irradiated NOD mice, allowing for reconsistution with a diabetogenic immune system. Steps for isolating pancreatic islet cells, which can then be used for a variety of purposes (e.g., as a source of islet antigens to establish and maintain autoreactive T cell lines) are included. Finally, steps are outlined that can be used to introduce transgenes into NOD mice. This protocol also discusses important considerations for introduction of targeted mutations produced in embryonic stem cells derived from other inbred strains, or introduction of other genes from non‐diabetes‐prone strains.