E boxes, recognition sequences for basic helix-loop-helix (bHLH) transcription factors, are detected in the enhancer and promoter regions of several murine type C retroviruses. Here we show that ALF1, a member of bHLH protein family of transcription factors, in vitro binds with differing affinities to distinct E-box sequences found in the U3 regulatory regions of Friend, Moloney, SL3-3, and Akv murine leukemia viruses (MLVs) as well as Friend spleen focus-forming virus (SFFV). In NIH 3T3 fibroblasts, ALF1 overexpression elevated transcription from the U3 region of Moloney MLV and the complete long terminal repeat regions of Friend SFFV, Akv MLV, and SL3-3 MLV but neither from the U3 region nor from the complete long terminal repeat of Friend MLV. Introduction of mutations in the Akv MLV E boxes showed the E- box cis elements to be required for the function of ALF1 as a transcription factor. ALF1 and the glucocorticoid receptor, with overlapping DNA binding sequences, did not act synergistically with respect to transcriptional trans activation of expression from the Akv MLV promoter-enhancer region. We conclude that ALF1 in vivo may be an important transcription regulator for Akv, SL3-3, and Moloney MLVs as well as for Friend SFFV.