AbstractThe VpreB1 protein is thought to be expressed on the surface of pre‐B cells in association with λ5 and μ, heavy chain, and to play an important role on B cell differentiation. The expression of VpreB1 and λ5 is pre‐B cell specific, and regulated at the initiation of transcription. We have identified at least two sequence‐specific DNA‐binding proteins which bind to the region −191 to ‐74 of the promoter of the mouse VpreB1 gene. These DNA‐binding proteins also bind to the promoter of the mouse λ5 gene. One of the two DNA‐binding proteins, called EBB‐1, is restricted to pre‐B and B cells, but not detected in plasma cells, T cells and cells of other lineages. Transient transfection analysis of reporter constructs revealed that the binding sites of these proteins play a significant role in the activity of the promoter, especially the binding site of EBB‐1. Taken together these results suggest that EBB‐1 might be one of the crucial factors which regulates a series of intracellular events in B cell differentiation.