From a human bone marrow cDNA library, we have cloned and sequenced a gene which cross-hybridized with murine pre-B cell-specific gene 8HS-20 cDNA under the low-stringent condition. Sequence analysis predicted that this gene (YM-1) encoded 240 amino acids which had the basic structure of immunoglobulin lambda light chain. The 3' half of the YM-1 sequence was identical to the J lambda 2 C lambda 2 region except for four nucleotides. The 5' part of the gene had 87.6% sequence homology with the reported V lambda gene called T1. Comparison of the deduced amino acid sequences with representative members of the seven other V lambda subgroups showed considerable structural homology, but the maximum homology with these chains was 44%. Therefore, we conclude that YM-1 belongs to a new V lambda subgroup. Interestingly YM-1 showed higher homology with VpreB1 (56%) than with any of the other V lambda subgroups. By Southern blot analysis four to six cross-hybridizing V lambda bands were detected at high stringency. Expression of the V lambda gene was observed in immature as well as mature B cell lines without accompanying V-JC gene joining, suggesting that V lambda of the YM-1 locus is activated at the early stage of maturation.