Recently, we have shown that the human immunodeficiency virus (HIV-1) long terminal repeat (LTR) directed chloramphenicol acetyltransferase (CAT) gene is efficiently expressed in human hepatoblastoma HepG2 cells and these cells can support productive HIV-1 replication. In this study we show that HepG2 cells contain a nuclear factor that binds to the HIV-1 trans-activating region (TAR), which we named HepG2-derived TAR binding protein (HTBP). Gel retardation assays using synthetic oligonucleotide probes carrying different mutations in the TAR region and competition DNA mobility-shift experiments using these oligonucleotides revealed the binding site encompassing between +7 and +13 nucleotides (5'-TCTGGTT-3') in the HIV-1 LTR. An in vivo CAT competition assay using -65HIV-1 LTR CAT as a reporter plasmid and various competitor plasmids containing these mutated oligonucleotides also demonstrated that HTBP can influence the HIV-1 LTR-directed CAT gene expression in HepG2 cells by interaction with a specific sequence in the TAR region.