There are several thyroid antigens including human sodium iodide symporter (hNIS), thyrotropin receptor (TSH-R), thyroid peroxidase (TPO), and thyroglobulin (Tg) that have been considered to be thyroid-specific proteins involved in the pathogenesis of autoimmune thyroid diseases. We examined the expression of these thyroid-tolerance related genes in normal human thymus, the lymphoid organ responsible for the induction of central T-cell self. Reverse transcription-polymerase chain reaction (RT-PCR) amplifications were performed with 4 pairs of oligonucleotide primers specific for the hNIS, TSH-R, TPO, and Tg genes, respectively. Gene-specific transcripts were confirmed by Southern hybridization using digoxigenin-labeled internal oligonucleotide probes. To monitor cDNA integrity and quantity, all samples were coamplified with a pair of intron-spanning human beta-actin-specific oligonucleotide primers. Furthermore, using a highly sensitive immunostaining technique and antibodies specific for these 4 antigens, we examined whether NIS-, TSH-R-, TPO-, and Tg-specific immunoreactivity can be detected and localized in normal human thymus. RT-PCR and Southern hybridization revealed expression of each of these 4 thyroid-related genes in normal human thymus. In addition, immunohistochemical analysis of frozen tissue sections derived from normal human thymus showed marked immunoreactivity for NIS, TSH-R, and Tg as well as weaker staining for TPO. Control reactions using isotype matched nonimmune immunoglobulins were consistently negative. Taken together, our results suggest that NIS-, TSH-R-, TPO-, and Tg-RNA are present and actively processed to immunoreactive NIS-, TSH-R-, TPO-, and Tg-like protein in human thymus. These data support the concept that pre-T lymphocytes may be educated to recognize thyroid-related epitopes expressed in thymus, and, thus, to generate self-tolerance against these thyroid-related antigens.