Several studies have provided indirect evidence in support of a role for β cell-specific Th2 cells in regulating insulin-dependent diabetes (IDDM). Whether a homogeneous population of Th2 cells having a defined β cell Ag specificity can prevent or suppress autoimmune diabetes is still unclear. In fact, recent studies have demonstrated that β cell-specific Th2 cell clones can induce IDDM. In this study we have established Th cell clones specific for glutamic acid decarboxylase 65 (GAD65), a known β cell autoantigen, from young unimmunized nonobese diabetic (NOD) mice. Adoptive transfer of a GAD65-specific Th2 cell clone (characterized by the secretion of IL-4, IL-5, and IL-10, but not IFN-γ or TGF-β) into 2-or 12-wk-old NOD female recipients prevented the progression of insulitis and subsequent development of overt IDDM. This prevention was marked by the establishment of a Th2-like cytokine profile in response to a panel of β cell autoantigens in cultures established from the spleen and pancreatic lymph nodes of recipient mice. The immunoregulatory function of a given Th cell clone was dependent on the relative levels of IFN-γ vs IL-4 and IL-10 secreted. These results provide direct evidence that β cell-specific Th2 cells can indeed prevent and suppress autoimmune diabetes in NOD mice.