The internal enhancer binding factor (IBF) that specifically binds sequences within the gag gene internal enhancer of Rous sarcoma virus Schmidt-Ruppin A was purified to near homogeneity from BHK cells. The polypeptides that constituted IBF DNA-binding activity were identified by sodium dodecyl sulfate-polyacrylamide gel analysis. As isolated from BHK cells, IBF consisted of two different but related polypeptides. One (IBF alpha) had a molecular weight of 40,000; the other (IBF beta) had a molecular weight of 20,000 and appeared to be a proteolytic product of IBF alpha. The site within the gag gene to which IBF bounds in vitro (internal enhancer site 2; nucleotides 856 to 878 of the Rous sarcoma virus genome) were demonstrated to function as a cis-acting transcriptional stimulatory element both in vivo and in vitro. By using HeLa cell nuclear transcription extracts, purified IBF was found to function as a trans-acting transcription factor that stimulated transcription in vitro. Purified IBF was also demonstrated to be very similar to EBP20 (K. Carlberg, T. A. Ryden, and K. Beemon, J. Virol. 62:1617-1624, 1988), and it may well belong to the same family of DNA-binding proteins.