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We have recently isolated cDNA clones representing four alternative splice forms of a T cell-specific transcription factor, TCF-1. Here we report the characterization of the human gene encoding this factor. The TCF-1 gene is contained in 10 exons including an untranslated first exon. The DNA-binding high mobility group (HMG) box of TCF-1 is encoded by the closely spaced exons VI and VII. Differential splicing involves an alternative exon (IX) and three splice acceptor sites in exon X. Based on comparison of sequence and on the placement of an alternative exon, TCF-1 appears closely related to the recently characterized HMG box transcription factor TCF-1 alpha/LEF. In particular, the HMG boxes encoded by the two TCF genes are virtually identical. The TCF-1 gene resides on chromosome 5 band q31.1. The TCF-1 promoter coincides with a CpG island. As determined by chloramphenicol acetyltransferase analysis, the promoter is preferentially active in T cells. The promoter does not contain TCF-1/TCF-1 alpha binding sites and is therefore not autoregulated. This observation implies the existence of yet uncharacterized T cell transcription factors that are active during early T cell differentiation. 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