Using gel mobility assays, we have detected two proteins that bind in the U3 region of the 1731 retrotransposon long terminal repeats (between positions -110 and -73) in nuclear extracts from Drosophila melanogaster cultured cells. The first one binds double-stranded DNA, whereas the other binds the mRNA-like strand in a sequence-specific manner. We report here the characterization of the latter protein, named NssBF for nuclear single-stranded-DNA binding factor. Gel filtration shows an apparent molecular mass of 95 kDa for NssBF. The points of contact between NssBF and its single-stranded DNA target were determined. This protein binds neither the complementary strand nor the corresponding RNA sequence. A possible role of NssBF in transcription is discussed.