A cDNA clone was isolated from a chicken embryo cDNA library employing a PCR-generated radiolabeled probe specific for the U3 region of the Rous sarcoma virus LTR. The cDNA encodes a protein of 345 aa and is homologous to the Y-box (inverted CCAAT) binding proteins. The amino acid sequence of the RSV-EF-1 shows 75% identity with rat EF1 but the NH₂-terminal 60-aa residues share little homology. At the carboxyl terminus an additional 28-aa sequence, rich in basic residues, probably encoded by an extra exon, is present in the chicken RSV-EF-1. Electrophoretic mobility assays carried out with various radiolabeled oligonucleotides spanning the U3 region of the RSV LTR (-234 to -54) indicated that the RSV-EF-1 binds strongly to the sequence AAGGTGGTAC and somewhat less efficiently to the sequences AAGGAAAG and CTTATGCAA. In contrast to rat EF1_A which binds to the inverted CCAAT box, RSV-EF-1 does not bind to the CCAAT box sequence. These results suggest that the RSV-EF-1, although structurally similar to the rat EF1_A, binds differently to more than one cis-acting element. The gene for RSV-EF-1 is expressed in a variety of cell lines, although most abundantly in avian fibroblasts compared to mammalian cells. It is barely expressed in normal livers but expressed at significantly enhanced levels in many immortalized hepatocytes and hepatic tumors.