Intrathymic expression of tissue-specific self-Ags can mediate tolerance of self-reactive T cells. However, in this study we define circumstances by which thymic expression of a tissue-specific autoepitope enhances positive selection of disease-causing, self-reactive T cells. An immunodominant gastritogenic epitope, namely the gastric H/K ATPase β subunit 253-277 (H/Kβ253-277), was attached to the C terminus of the invariant chain (Ii) and the hybrid Ii (Ii-H/Kβ 253-277) expressed in mice under control of the Ii promoter. The Ii-H/Kβ253-277 fusion protein was localized to MHC class II-expressing cells in the thymus and periphery of Ii-H/Kβ 253-277 transgenic mice. In one transgenic line the level of presentation in the periphery (spleen) was insufficient to activate naive, low affinity H/Kβ253-277-specific transgenic T cells (1E4-TCR), whereas thymic presentation of H/Kβ253-277 enhanced positive selection of 1E4-TCR cells in Ii-H/Kβ253-277/1E4-TCR double-transgenic mice. Furthermore, Ii-H/Kβ253-277/1E4-TCR double-transgenic mice had an increased incidence of autoimmune gastritis compared with 1E4-TCR single-transgenic mice, demonstrating that the 1E4 T cells that seeded the periphery of Ii-H/Kβ253-277 mice were pathogenic. Therefore, low levels of tissue-specific Ags in the thymus can result in positive selection of low avidity, self-reactive T cells. These findings also suggest that the precise level of tissue-specific Ags in the thymus may be an important consideration in protection against autoimmune disease and that perturbation of the levels of self-Ags may be detrimental.