A region of the human T-cell leukemia virus long terminal repeat (HTLV-I LTR) located between -155 and -117 is important in the regulation of gene expression by the ets family of transcription factors. In an attempt to identify additional cellular transcription factors that bind to this portion of the HTLV-I LTR, we used lambda gt11 expression cloning with oligonucleotides corresponding to this element. A 1239-bp cDNA was isolated from a Jurkat cDNA library, which encoded a protein capable of binding to this purine-rich region. This protein, which we designated human T-cell leukemia virus enhancer factor (HTLF), contains a domain with homology to the recently described fork head DNA binding domain. Chromosome mapping of the HTLF gene demonstrated that it was localized to human chromosome 2p16-p22. HTLF is a unique cellular gene that may function in the transcriptional regulation of HTLV-I LTR.