The avian leukosis and sarcoma virus long terminal repeat (LTR) enhancers feature directly repeated CCAAT/enhancer element sequences which are also found in many viral and cellular gene enhancers. While most members of the CCAAT/enhancer element-binding protein (C/EBP) transcription factor family exhibit tissue-restricted expression, there may be ubiquitously expressed C/EBP-like factors that regulate widespread CCAAT/enhancer element-driven transcription. An avian C/EBP-related factor designated Al/EBP was previ- ously shown to bind CCAAT/enhancer elements within the avian leukosis virus (ALV) and Rous sarcoma virus (RSV) LTR enhancers in a pattern identical to that of a B-cell LTR-binding factor (W. J. Bowers and A. Ruddell, J. Virol. 66:6578-6586, 1992). An Al/EBP-specific antiserum recognizes a 40-kDa LTR CCAAT/enhancer element-binding protein purified from avian B lymphoma cells. A1/EBP is widely expressed at the mRNA and protein levels, suggesting that this protein could be important not only in regulating widespread expression of the AIN and RSV retroviruses but also in controlling the expression of other viral and cellular gene enhancers that possess CCAAT/enhancer motifs. We also found that an NF-KB/Rel-related protein is a component of the LTR CCAAT/enhancer element binding complex through its interaction with A1/EBP. At least one of the NF-kappaB family members, p65 (RelA), is capable of activating LTR CCAAT/enhancer element-driven transcription. These findings suggest a role for Rel-related factors in the regulation of AIN or RSV LTR-driven transcription via an interaction with Al/EBP.