The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.Viruses have proven to be excellent models for eucaryotic transcription because of their relatively small size and ease of genetic manipulation. The existence of important cisacting regulatory regions such as enhancers (16), as well as the nature of the few well-characterized mammalian mRNA transcription factors, was discovered by studying viral gene regulation (1,4,22). The enhancer element of the gibbon ape leukemia virus (GALV) is suitable for such a study. Holbrook et al. (15) recently demonstrated that the GALV enhancer element is active in several uninfected cell lines. We have sought to identify the cellular products which trans-activate this enhancer. The enhancer activity of the GALV Seato strain has been shown to reside in a 155-basepair (bp) region within the U3 region of the long terminal repeat (LTR). The enhancer element is bounded by Apal and BanI restriction endonuclease cleavage sites and includes tandem, direct, and perfect repeats of a 48-bp sequence (15).Several viral variants of the GALV enhancer element exist which exhibit different levels of enhancer activity in vivo. The GALV Seato and San Francisco strains possess a homologous element which displays strong enhancer activity in a variety of cell types (15). The GALV San Francisco strain contains only one of the repeated 48-bp sequences (27). These strains are associated with different hematopoietic malignancies in primates; GALV Seato is associated with granulocytic leukemia, and GALV San Francisco with lymphocytic leukemia (27). There is also a GALV LTR variant (CM9), which was recovered from the GALVcontaining, transformed gibbon T-cell line MLA144 (3). This variant contains a 94-bp direct repeat which has very weak but detectable enhancer activity (N. Holbrook, A. Gulino, ...