The Bovine Leukemia Virus (BLV) promoter is located in its 5' Long Terminal Repeat (LTR), composed of the U3, R and U5 regions. BLV transcription is regulated by cis-acting elements located in the U3 region, including three 21-bp enhancers required for transactivation of the BLV promoter by the virus-encoded transactivator TaxBLV. In addition to the U3 cis-acting elements, both the R and U5 regions contain stimulatory sequences. To date, no transcription factor binding site has been identified in the R region. Here, sequence analysis of this region revealed the presence of a potential E-box motif (5'-CACGTG-3'). By competition and supershift gel shift assays, we demonstrated that the basic-helix-loop-helix transcription factors USF1 and USF2 specifically interacted with this R region E-box motif. Mutations abolishing USF binding caused a reproducible decrease in basal or Tax-activated BLV promoter-driven gene expression in transient transfection assays of B-lymphoid cell lines. Cotransfection experiments showed that the USF1 and USF2a transactivators were able to act through the BLV R region E-box. Moreover, combined mutation of both this E-box motif and of the Interferon Regulatory Factor site previously identified in U5 decreased the LTR basal activity to a greater degree than the individual mutations. Taken together, these results physically and functionally characterize an USF-binding site in the R region of BLV. This E-box motif located downstream of the transcription start site constitutes a new positive regulatory element involved in the transcriptional activity of the BLV promoter and could play an important role in virus replication.