The long terminal repeats (LTRs) of RNA tumor viruses, including human T-cell lymphotropic virus type I (HTLV-I), contain the control elements for expression of viral genes. Sequence-specific LTR-DNA-binding proteins could regulate viral functions. To search for such proteins we have used an in vitro non-denaturing polyacrylamide gel assay, with restriction fragments of the HTLV-I LTR and nuclear protein extracts from several HTLV-I-infected cell lines and an uninfected T-cell line, H9. Four DNA-binding activities were observed, including non-specific DNA-binding activity and at least two activities (forms I and II) which bind specifically to a HinfI restriction fragment from nucleotides +181 to +334 relative to the transcription start site. DNA-binding activities I and II were partially resolved by ion-exchange chromatography and mapped by protection experiments to two 10-20-bp blocks surrounding the polyadenylation site at +221. Of the cell lines tested, form II was abundantly found in C10/MJ, and forms I and IV were also found in C91/PL, C81-66-45, MT2 and H9 cells.