The BP-1/6C3 antigen is a phosphorylated cell surface glycoprotein that can be identified by monoclonal antibodies on mouse pre-B cells, immature B cells, and certain stromal cell lines from bone marrow. Expression of this antigen is increased in stromal-dependent pre-B cell lines and retrovirally transformed pre-B cells. Expression of the BP-1/6C3 antigen thus correlates with proliferation and transformation of immature B-lineage cells. In this study, we report the isolation and characterization of cDNAs encoding the BP-1/6C3 antigen. Northern blot analysis revealed a major 4.1-kilobase mRNA in all BP-1/6C3+ mouse pre-B lines and in a human pre-B cell line. BP-1/6C3 mRNA was either absent or truncated in BP-1/6C3- cell lines. The cDNA sequence predicts a type II integral membrane protein of 945 amino acids with an intracytoplasmic amino terminus of only 17 amino acids and a typical zinc-binding motif in its extracellular domain. BP-1/6C3 has significant homology to aminopeptidase N and is the second member of the zinc-dependent metallopeptidase gene family to be found on the surface of early B-lineage cells.