Human lambda light chains of the recently recognized variable region (VL) subgroup V lambda VIII can be distinguished from proteins of other V lambda gene families on the basis of distinctive chemical and serologic properties and by their preferential association with certain types of autoantibodies, i.e. rheumatoid factors (RFs). We now report that we have cloned from a human placental library a V lambda VIII-encoding germline gene, designated IGLV8A1, using as a molecular probe a partial V lambda VIII fragment generated by polymerase chain reaction (PCR) from genomic DNA. IGLV8A1 contained all the requisite elements of a potentially functional gene, including a V lambda exon with an open reading frame encoding 103 residues. Its expressed products were identified through analyses of cDNA cloned from two different monoclonal lambda VIII B-cell populations. The primary structure of lambda VIII light chains differed from that of lambda I, lambda II, lambda III, lambda IV and lambda VI proteins by the presence of distinctive residues within the first framework region (FR1) and an 11- rather than 7-residue second complementarity-determining region (CDR2). Remarkably, the IGLV8A1 gene was more homologous to the two functional rabbit V lambda germline genes, RV lambda 2 and RV lambda 3 (including the presence of one extra codon within the leader sequence), and to the murine V lambda x gene. Light chains encoded by the human, rabbit and mouse lambda VIII-related genes shared certain unique primary structural features, notably the four additional CDR2 residues. The evolutionary conserved nature of the human V lambda gene and, in particular, the apparently novel tertiary structural effects induced by an elongated CDR2 provide evidence for the biological and functional importance of the V lambda VIII subgroup.