We define a protein complex present in avian nuclear extracts that interacts with the Schmidt-Ruppin strain of the Rous sarcoma virus (RSV) long terminal repeat (LTR) between positions -197 and -168 relative to the transcriptional start site. We call this complex EFIV and demonstrate that the EFIV protein(s) is present in several avian cell types examined, including B cells (S13 and DT40), T cells (MSB), and chicken embryo fibroblasts. We also report that the EFIV binding site activates transcription of reporter constructs after transfection into avian B cells and chicken embryo fibroblasts, demonstrating that the EFIV region constitutes a functional transactivator sequence. By chemical interference footprinting and mutational analyses we define the EFIV binding site as including the sequence GCAACATG, which is present in two copies between positions - 197 and -168, as well as sequences that lie between the two repeats. Electrophoretic mobility shift competition experiments suggest that the EFFV protein(s) may be related to members of the CCAAT/enhancerbinding protein family of transcription factors that interact with different regions of the RSV and the avian leukosis virus (ALV) LTRs. However, as defined by differences in sensitivity to protein synthesis inhibitors and footprinting patterns, EFIV is clearly distinct from these previously defined LTR binding factors. In addition, the finding that EFTV binding activity is stable in B cells indicates either that the lability of all 5′ LTR binding activities is not required for B-cell transformation by the ALV/RSV family of viruses or that nonacute transforming viruses that include an RSV LTR may use a mechanism to effect cellular transformation different from that proposed for ALV.