The HTLV-I provirus contains two different promoters: the classical retroviral promoter in the 5' long terminal repeat (LTR) and our previously identified second promoter in the pol gene just upstream of the ATG codon of the tax gene. Here, we demonstrated that the internal promoter expresses the gene for Tax but not Rex. As the deletion of upstream of the transcriptional initiation site (nt 5130) caused down-regulation of the promoter activity, we termed the region HTLV-I internal regulatory element (HIRE). We found a cellular sequence-specific DNA binding protein which binds to HIRE. Furthermore, we demonstrated that the 3' LTR regulates Tax expression from the internal promoter. These findings may shed light on a novel mechanism for gene expression in complex retroviruses of the HTLV family.