OBJECTIVE The expression of tissue-specific self-antigens in the thymus is essential for self-tolerance. Genetic susceptibility to type 1 diabetes correlates inversely with thymic insulin expression and, in mice, lowered levels of this expression result in T cell responses against insulin. This study was undertaken to examine whether thymic insulin expression is regulated by the same metabolic stimuli as in beta cells or by different inputs, possibly of an immune nature. METHODS Ins2 mRNA changes in mouse thymus were evaluated in vivo, following intraperitoneal glucose injection. We also examined the effect of a high glucose concentration on Ins2 mRNA in clones of insulin-expressing medullary thymus epithelial cell lines (mTECs). The same in vitro system was used to evaluate the effect of IFN-gamma and cell-to-cell contact with thymocytes in co-culture. RESULTS Ins2 mRNA was significantly increased in the pancreas following a glucose load, but remained unchanged in the thymus. Furthermore, stimulation of insulin-expressing mTECs in vitro with IFN-gamma, a cytokine involved in T cell negative selection, decreased levels of insulin expression even though expression of Aire was increased. Last, co-culture of mTECs with thymocytes resulted in an upregulation of both Aire and insulin expression. CONCLUSIONS We conclude that regulation of insulin transcription in the thymus is not dependent on metabolic stimuli but it may, instead, be under the control of cytokines and cell-to-cell interactions with lymphoid cells. That this regulation is not always coordinated with that of Aire, a non-specific master switch, suggests insulin-specific mechanisms.