Through extensive serologic, chemical, and molecular studies involving monoclonal Ig proteins and B cell-related populations, we provide definitive evidence that the V lambda IV subgroup of human light (L) chains is separate and distinct from V lambda III and all other known V lambda gene families. lambda IV and lambda III L chains were differentiated immunochemically using well-characterized polyclonal and monoclonal anti-V lambda subgroup-specific Abs. Prototypic L chains, originally classified as lambda IV on the basis of distinctive framework region 1 residues, were distinguished serologically from lambda IIIa, lambda IIIb, and lambda IIIc proteins and also from lambda I, lambda II, lambda VI, and lambda VIII L chains. Furthermore, by using anti-lambda IV reagents, we identified eight additional monoclonal V lambda IV-related populations, including three IgM rheumatoid factor-producing cell lines. The percentage of homology among the lambda IV proteins ranged from 83 to 100, vs 53 to 72 when compared with lambda III components. Moreover, lambda IV proteins shared particular subgroup-associated FR and complementarity-determining region residues. At the molecular level, the nucleotide sequences encoding two of the IgM lambda IV rheumatoid factors were identical to that found for the genomic counterpart, as well as to the previously reported IGLV3S1 and Humlv418 germ-line genes. Two other lambda IV cDNAs contained additional non-germ-line-encoded nucleotides at the V-J joint. The single or pauci-gene nature of the V lambda IV family was evidenced from Southern blotting and from the extensive sequence homology among lambda IV components. Our studies have provided further evidence for the prevalence of lambda IV L chains among Ig lambda autoantibodies, thus implying a functional significance for the V lambda IV subgroup.