Administration of antigens via mucosal routes, such as orally or intranasally, can induce specific immunological tolerance and has been used as a rational basis for the treatment of autoimmune diseases, including type 1 diabetes. Recently, however, orally delivered antigens were shown to induce CD8 cytotoxic T-lymphocytes (CTLs) capable of causing autoimmune diabetes. In this report, we have examined several mucosal routes for their ability to induce CTLs and autoimmune diabetes, with the aim of identifying approaches that would maximize tolerance and minimize CTL generation. In normal C57BL/6 mice, ovalbumin (OVA) delivered by either the oral or nasal routes or by aerosol inhalation was able to prime CTL immunity in both high- and low-dose regimens. To address the relevance of these CTLs to autoimmune disease, OVA was given to mice that transgenically expressed this antigen in their pancreatic β-cells. Irrespective of antigen dose or the route of delivery, mucosal OVA triggered diabetes, particularly after intranasal administration. These findings suggest that CTL immunity is likely to be a consequence of mucosal antigen delivery, regardless of the regimen, and should be considered in the clinical application of mucosal tolerance to autoimmune disease prevention.