The specificity of polyclonal mouse rheumatoid factors (RF) was analyzed by competition experiments with heat-aggregated mouse IgG subclasses. The RF spontaneously produced by three normal mouse strains (129/Sv, CBA/Ht, and C57Bl/6) and by two strains with autoimmune diseases (MRL/l and NZB) were found to consist of distinct non-cross-reactive antibody subpopulations each specific for one IgG subclass. The sera of the normal strains contained IgG1- and IgG2a-specific RF. The autoimmune strains produced an additional variety of RF that was specific for The autoimmune strains produced an additional variety of RF that was specific for IgG2b. Also, the RF secreted by spleen cells of various normal strains after in vitro polyclonal activation with lipopolysaccharide could be resolved into distinct subpopulations specific for IgG1 or IgG2a. These results were confirmed by the analysis of monoclonal RF derived from BALB/c, C57Bl/6, CBA/Ht, and 129/Sv mice: of 73 hybridomas with RF activity, 71 displayed a strict subclass specificity. The subclass predominantly recognized depended on the origin of the spleen cells used to generate the hybridomas. After polyclonal activation in vitro, a broad spectrum of different specificities was obtained with 16 RF specific for IgG1, 13 for IgG2a, and 4 for IgG2b. In contrast, 27 of 28 monoclonal RF derived from 129/Sv and BALB/c mice without prior polyclonal activation were specific for IgG2a, and of these 75% were allotype specific since they failed to react with IgG2a of the b allotype. These results demonstrate the importance of subclass specificity in the production of RF in vivo. With the exception of the IgG2b-specific clones, all these monoclonal RF reacted preferentially with heat-aggregated or antigen-bound IgG. Among the hybridomas generated by the fusion of in vitro polyclonally activated spleen cells of 4-wk-old mice, the frequency of clones with RF activity was at least 40 times higher than that of clones specific for mouse IgM, human IgG, ovalbumin, and hen lysozyme.