Regulatory anti-diabetogenic T cells (T(reg)) can be induced by the mucosal administration of insulin or proinsulin peptides, in the non-obese diabetic (NOD) mouse model of autoimmune type 1 diabetes. Naso-respirtory insulin (which avoids insulin degradation) induces CD8+ alpha(alpha) TCR gamma(delta) T(reg) whereas peptides that bind to the NOD MHC class II molecule, I-Ag7, insulin B9-23 and proinsulin B24-C36, induce CD4+ T(regs) Following naso-respiratory delivery of insulin to NOD mice increased numbers of CD8+ gamma(delta) T cells expressing interleukin (IL)10 are detected in the pancreatic lymph nodes. Neonatal (3 day) thymectomy (NTX) dramatically accelerates diabetes development in NOD mice, associated with lymphopaenia and a block in the maturation of mucosal intrepithelial lymphocytes (IEL), especially extrathymic-derived CD8+ alpha(alpha) TCR gamma(delta) IEL. Regulatory anti-diabetogenic T cells cannot be elicited by naso-respiratory insulin in NTX-NOD mice. Reconstitution of NTX-NOD mice with CD8+ alpha(alpha) TCR gamma(delta) T cells prevents diabetes. CD8+ gamma(delta) T(reg) are conceivably physiological and insulin-specific, induced by exposure to insulin in maternal milk. These findings infer an immunoregulatory role for extrathymic-derived IEL, developing under the influence of the thymus and conditioned by early exposure to the exogenous environment.