Abstract “Promiscuous” thymic expression of peripheral autoantigens can contribute to immunological tolerance in some cases. However, in this study we show that thymic mRNA expression alone cannot predict a contribution to thymic tolerance. Autoimmune gastritis is caused by CD4+ T cells directed to the α (H/Kα) and β (H/Kβ) subunits of the gastric membrane protein the H+/K+ ATPase. H/Kα mRNA is expressed in the thymus, but H/Kβ expression is barely detectable. In this study, we demonstrate that thymic H/Kα in wild-type mice or mice that overexpressed H/Kα did not result in negative selection of pathogenic anti-H/Kα T cells. However, negative selection of anti-H/Kα T cells did occur if H/Kβ was artificially overexpressed in the thymus. Given that H/Kα cannot be exported from the endoplasmic reticulum and is rapidly degraded in the absence of H/Kβ, we conclude that H/Kα epitopes are unable to access MHC class II loading compartments in cells of the normal thymus. This work, taken together with our previous studies, highlights that thymic autoantigen expression does not necessarily result in the induction of tolerance.