The V(preB1) protein is thought to be expressed on the surface of pre-B cells in association with lambda(5) and mu-heavy chain, and to play an important role on B cell differentiation. The expression of V(preB1) and lambda(5) 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 V(preB1) gene. These DNA-binding proteins also bind to the promoter of the mouse lambda(5) 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.