Background: In rheumatoid arthritis (RA), B-lineage cells in the synovial membrane secrete large amounts of immunoglobulin that contribute to tissue destruction. The CDR3 of an immunoglobulin light chain is formed by rearrangements of V(L) and J(L) gene segments. Addition of non-germline- encoded (N) nucleotides at V(D)J joins by the enzyme terminal deoxynucleotidyl transferase (TdT) enhances antibody diversity. TdT was previously thought to be active in B cells only during heavy chain rearrangement, but we and others reported unexpectedly high levels of N addition in kappa light chains. We also found clonally related kappa chains bearing unusually long CDR3 intervals in RA synovium, suggesting oligoclonal expansion of a set of atypical B lymphocytes. In this study, we analyzed lambda light chain expression to determine if N addition occurs throughout immunoglobulin gene rearrangement and to compare CDR3 lengths of lambda and kappa light chains in RA patients and normal individuals. Materials and Methods: Reverse transcription-polymerase chain reaction (RT-PCR) amplification of VλIII transcripts was performed on RA synovia and peripheral blood lymphocytes (PBL) and normal PBL for which kappa repertoires were previously analyzed. Representative ;λ+ PCR products were cloned and sequenced. Results: Analysis of 161 cDNA clones revealed that N addition occurs in lambda light chains of RA patients and normal controls. The lambda light chain repertoires in RA were enriched for long CDR3 intervals. In both RA and controls, CDR3 lengths were strongly influenced by which Vλ gene segment was present in the rearrangement. Five sets of clonally related sequences were found in RA synovia and PBL; one set was found in normal PBL. Conclusions: In humans, unlike mice, N addition enhances antibody diversity at all stages of immunoglobulin assembly, and the structural diversity of lambda CDR3 intervals is greater than that of kappa light chains. Clonally related Vλ gene segments in RA support an antigen-driven B-cell response.