We have cloned a cDNA (p3C4 lambda 5) encoding a human immunoglobulin lambda-chain of human-mouse heterohybridoma, H6-3C4, which produces a human monoclonal antibody against human sperm surface antigen. Amino acid sequence deduced from the nucleotide sequence of V lambda (V lambda 3C4) of cloned cDNA was closely related to that of SHV lambda protein, a member of subgroup IV. Southern blot analysis of human genomic DNAs by using V lambda 3C4 as a probe detected at least 8 cross-hybridizing members of subgroup V lambda IV. Restriction enzyme fragment polymorphism (RFLP) appeared to be present in normal individuals with V lambda 3C4 gene.