The enhancer region of Akv murine leukemia virus contains the sequence motif ACAGAIGG. This sequence is homologous to the E-box motif originally defined as a regulatory element in the enhancers of immunoglobulin μ and κ genes. We have used double-stranded oligonucleotide probes, corresponding to the E box of the murine leukemia virus Akv, to screen a randomly primed λgt11 cDNA expression library made from mouse NIH 3T3 fibroblast RNA. We have identified seven λ clones expressing DNA-binding proteins representing two different genes termed ALF1 and ALF2. The results of sequencing ALF2 cDNA suggests that we have recovered the gene for the basic-helix-loop-helix transcription factor Al, the murine analog of the human transcription factor E47. The cDNA sequence of ALF1 codes for a new member of the basic-helix-loop-helix protein family. Two splice variants of ALF1 cDNA have been found, differing by a 72-bp insertion, coding for putative proteins of 682 and 706 amino acids. The two ALF1 mRNAs are expressed at various levels in mouse tissues. In vitro DNA binding assays, using prokaryotically expressed ALF1 proteins, demonstrated specific binding of the ALF1 proteins to the Akv murine leukemia virus E-box motif ACAGATGG. Expression in NIH 3T3 fibroblasts of GAL4-ALF1 chimeric protein stimulated expression from a minimal promoter linked to a GAL4 binding site, indicating the existence of a transcriptional activator domain in ALF1.