The regulation of gene expression in thymic epithelial cells is critical for T cell development. The mouse thymic epithelial gene Tscot encodes a protein with weak homology to bacterial 12 transmembrane co-transporters. Using competitive reverse transcription-polymerase chain reaction (RT-PCR), we show that low level Tscot expression is detectable in several other tissues. Tscot was mapped to chromosome 4 and was also detected in other mammalian species by Southern blotting. The human cDNA clone showed 77% amino acid identity with the mouse sequence. The highest conservation was in the TM regions and in a small segment of the central cytoplasmic loop. Genomic clones spanning 17164 bases of the Tscot gene revealed four exons with nine of the TM domains encoded in the first exon. The major transcriptional start site in mouse was identified by a primer extension analysis and confirmed by RT-PCR. Comparison of 1.7 kb of the human and mouse promoters identified six conserved possible regulatory elements, one containing a potential binding site for an interferon alpha inducible factor. Finally, as a functional test, 3 kb of the murine promoter was used to create a transgenic mouse that expresses enhanced green fluorescent protein message strongly in the thymus, weakly in the kidney and undetectably in the spleen, liver and heart.