The Epstein-Barr virus gene BRLF1 encodes the transcription factor R, which is a sequence-specific DNA-binding protein important for the switch from latency to a productive cycle. We have defined a repertoire of specific R-binding sites using a GST-R fusion protein and a pool of 23 bp random DNA sequences. The R-bound sequences were selected by several rounds of Electrophoretic Mobility Shift Assay (EMSA) and amplification by PCR. Among the 45 sites selected, some positions in the sequences were highly conserved, i.e., 5'-GTGCC N7GTGGTG-3'. The guanine methylation assay revealed that R simultaneously contacts guanines in the two conserved cores, defining the consensus binding site 5'-GNCC N9 GGNG-3', and 30 sites among the 45 selected have this sequence. This last result also suggests that R binds two adjacent major grooves of the DNA. As shown by EMSA assay, R binds to all the sites tested with a comparable affinity, and they all mediate R-induced transcriptional activation in a transient expression assay.