To determine the molecular and functional properties of human rheumatoid factors (RF), we established stable hybridomas and Epstein-Barr virus-transformed B cell lines from the synovial fluid or peripheral blood of three patients with rheumatoid arthritis and one patient with systemic lupus erythematosus. 17 cell lines were obtained that produced high-titer immunoglobulin M (IgM) RF that reacted exclusively with rabbit but not human IgG or IgG of other mammalian species. Certain anti-rabbit IgG RF also had specificity for other mammalian antigens (Ag), including cytoskeletal proteins and intracellular proteins found in HeLa cells, as well as for Ag present in an extract prepared from the cell wall of group A streptococci. 13 of the 17 RF contained h-type light (L) chains, of which 12 were classified serologically as members of the γ-L chain variable region (VH) subgroup, designated VγIII. The heavy chain V region (VH) and Vx sequences of nine of these IgMX tLF were determined at the cDNA level. Five VH genes in three V. families were used by these antibodies (Ab), including VH1(dp21/1-4b and dpl0 [51pl]/ hv1051), VH3 (dp38/3-15 and dp77/13-21), and VH4 (dp70/4-4b). The deduced V gene-encoded amino acid sequences of the X chains of these IgMX tLF confirmed their serological classification as γIII, and they were further classified as members of the relatively uncommon VγIII subgroup, designated VγIIIb. Based on cDNA analyses, nine were the product of three different VγIIIb germline genes. Two such genes, designated hsiggl1150 and hsiggl1295, were cloned and sequenced from genomic DNA. Unique combinations of these V. and VγIIIb genes could be related to distinctive patterns of reactivity among the IgMγ RF. Although the VH and Vγ regions of these Abs were expressed primarily as germline-encoded sequences, four of nine multireactive Abs had extensive V region mutation, indicative of an Ag-driven process. The finding that γIIIb L chains are preferentially found among anti-rabbit IgG RF, and that some of these Ab have specificity for other protein, cellular, and bacterial Ag, provides new insight into the pathogenesis of RA and related diseases. © 1994, Rockefeller University Press., All rights reserved.