Induction of T cell tolerance in the thymus (central tolerance) is essential for preventing organ-specific autoimmunity. This apparent paradox is in part explained by promiscuous expression of numerous tissue-restricted self-antigens (TRA) in medullary thymic epithelial cells (mTEC), which is highly conserved between mice and man. In animal models, the threshold of central tolerance towards such TRA is surprisingly sensitive towards minor shifts in antigen expression levels and this might also be the case in humans. To precisely assess the inter-individual variability of TRA expression in man, we determined the level of transcription of several auto-antigens in purified human mTEC and subsets thereof by quantitative RT-PCR. We detected two expression patterns: first, high variability (>20-fold) correlated with autoimmune regulator (Aire) expression and mTEC differentiation, and secondly, non-correlated low variability. Importantly, our approach revealed a significantly higher Aire-correlated insulin transcription in mTEC of carriers of the protective insulin-dependent diabetes mellitus locus 2 haplotype compared to the non-protective haplotype. The considerable, yet selective variability in thymic expression levels of target auto-antigen expression might constitute a hitherto underestimated risk factor for the susceptibility of autoimmune diseases in man.