We have previously reported on the cloning and characterization of chk-YB-2, a novel member of the Y-box family of proteins, that binds to the sequence 5'-GTACCACC-3' present on the noncoding strand of the Rous sarcoma virus (RSV) long terminal repeat (LTR) in a single-strand-specific manner. Here, we demonstrate that deletion or mutation of this motif not only eliminates chk-YB-2 binding in vitro but also down-regulates RSV LTR-driven transcription in avian cells. Selective abrogation of chk-YB-2 expression by using antisense oligonucleotides decreased RSV LTR-driven transcription in a promoter-specific manner. This inhibition was not observed when a reporter construct with a deletion in the chk-YB-2 binding site was used. Depletion of cellular chk-YB-2 by transfecting the cells with excess of its recognition sequence oligonucleotides also resulted in reduced transcription from the RSV LTR. Taken together, these results suggest that chk-YB-2 acts as an activator of LTR-promoted transcription in avian cells and that this activation is mediated primarily through the sequence 5'-GTACCACC-3'.