Graves' ophthalmopathy, a human autoimmune disease of unknown etiology, is strongly associated with autoimmune hyperthyroidism. A major controversy is whether retro-ocular muscle or orbital fat/connective tissue is the target of the immune response. Previously, we observed preferential PCR amplification of lambda (relative to kappa) light chain DNA from cDNA of Graves' orbital tissue-infiltrating B cells/plasma cells. There is little information on V lambda gene usage in man and none in diseased tissue. To characterize the orbital lambda light chains, we constructed cDNA libraries using PCR-amplified DNA from three tissues and sequenced the variable region genes from randomly selected clones. Analysis of 27 clones from orbital fat/connective tissue libraries from two patients with acute inflammatory eye disease, and 15 clones from orbital muscle of one of these patients, revealed a diverse spectrum of lambda V region genes. The nucleotide sequences of these 42 clones were most homologous to 12 different germline genes: four family I (subfamilies I-a, -b and -c), three family II, two family III and one family VII germline genes. Each orbital tissue had a distinct profile of V lambda sequences. However, all clones used J lambda 2/3 and all three orbital tissues contained clones related to family II genes. Although some clones had V region sequences in near germline conformation, the majority differed from the closest germline gene in both framework and complementarity determining regions. Whether or not these differences result from multiple germline gene usage or somatic mutation of a smaller number of germline genes cannot be determined until information on the V lambda repertoire and its polymorphisms is complete. However, the V lambda gene diversity we observed in both orbital muscle and orbital fat/connective tissue suggests a role for lambda autoantibodies in the pathogenesis of Graves' ophthalmopathy.