Proinsulin, like many tissue-specific antigens, is expressed by rare (1-3%) cells of the thymus medullary stroma, presumably for the purpose of self-tolerance. Levels of this expression are associated with type 1 diabetes susceptibility in humans and in the NOD mouse. To further understand the mechanism of central tolerance induction by these rare cells, we have isolated and cultured two proinsulin-producing epithelial cell clones from murine thymus. These cells have a typical epithelial morphology and, by flow cytometry, a surface phenotype representative of mature thymic medullary epithelial cells (G8.8(+)/UEA-1(+)/DEC205(-)/CD45(-)/MHC II(+)). By RT-PCR, they express predominantly Ins2 as opposed to Ins1, as does whole thymus. Expression of the transcription factor Aire, implicated in enhancing promiscuous thymic expression of tissue-specific antigens, fell to very low levels after a few passages but increased 20-fold upon exposure to an agonistic anti-lymphotoxin B antibody, concurrent with 2.5-fold enhanced insulin expression. RNA of Pdx-1, Glut-2, and Gck was detectable by RT-PCR in whole thymus but not in the clones, suggesting thymic proinsulin expression is Pdx-1 independent and that Pdx-1, Glut-2, and Gck are likely expressed in the thymus as antigens, not as regulatory molecules.