During T cell differentiation, medullary thymic epithelial cells (MTEC) expose developing T cells to tissue-specific antigens. MTEC expression of such self-antigens requires the transcription factor autoimmune regulator (Aire). In mammals, defects in aire result in multi-tissue, T cell-mediated autoimmunity. Because the T cell receptor repertoire is randomly generated and extremely diverse in all jawed vertebrates, it is likely that an aire-dependent T cell tolerance mechanism also exists in nonmammalian vertebrates. We have isolated aire genes from animals in all gnathostome classes except the cartilaginous fish by a combination of molecular techniques and scanning of expressed sequence tags and genomic databases. The deduced amino acid sequences of Aire were compared among mouse, human, opossum, chicken, Xenopus, zebrafish, and pufferfish. The first of two plant homeodomains (PHD) in human Aire and regions associated with nuclear and cytoplasmic localization are evolutionarily conserved, while other domains are either absent or divergent in one or more vertebrate classes. Furthermore, the second zinc-binding domain previously named Aire PHD2 appears to have greater sequence similarity with Ring finger domains than to PHD domains. Point mutations in defective human aire genes are generally found in the most evolutionarily conserved regions of the protein. These findings reveal a very rapid evolution of certain regions of aire during vertebrate evolution and support the existence of an aire-dependent mechanism of T cell tolerance dating back at least to the emergence of bony fish.