The viral US3 and US6 gene products of human cytomegalovirus (CMV) are sequentially expressed at immediate-early and early times after infection, respectively. They downregulate the surface expression of HLA class I molecules. There are two repeat-containing regulatory regions between the US3 promoter and the US6 transcription unit designated R1 and R2. R2 contains repetitions of the NF-kappa B responsive element and enhances the immediate-early expression of the US3 gene. R1 contains 19 repetitions of a 5'-TRTCG-3' pentanucleotide arranged as everted repeats, inverted repeats, and variably spaced single pentanucleotides. In the context of the viral genome, R1 also enhances immediate-early US3 gene expression by an unknown mechanism (G. C. Bullock, et al., 2001, Virology 288, 164-174). We report a sequence motif within the R1 element that binds a human cell nuclear protein which is antigenically related to the Drosophila boundary element-associated factor (BEAF). The potential role of a 35-kDa cellular protein that binds to sequence motifs within the R1 element in regulating the expression of the CMV US3 immune evasion gene is discussed.