MRL/lpr/lpr (MRL/l) mice develop a lupus-like syndrome and a disease histologically and serologically similar to human rheumatoid arthritis. Their sera contain polyclonal IgM rheumatoid factors (RF) reactive with all murine IgG subclasses (frequently strongest with IgG2a) and several heterologous IgG. To examine the repertoire and epitopic specificities of these RF, we fused splenocytes from 3.5-mo-old seropositive MRL/l mice with appropriate myeloma partners and derived 1,723 hybridomas of which 23 secreted IgMRF. These monoclonal IgMRF bound to murine IgG only, not to other murine isotypes. Eight murine IgG subclass-specific clonotypes were identified. Most clones reacted with either multiple IgG subclasses or with IgG2a alone. A few clones reacted solely with IgG2b but none reacted exclusively with IgG1 or IgG3. Monoclonal IgMRF with exclusively anti-IgG2a activity exhibited allotypic specificity, reacting, with few exceptions, with a, c, and e, but not b, d, or j IgG2a allotypes. Four clonotypes could be distinguished by cross-reactivity with IgG from species other than mice. Monoclonals possessing activity against several murine subclasses cross-reacted extensively with heterologous IgG, including all human IgG subclasses without allotypic restrictions. Monoclonal IgMRF specific for murine IgG2a or 2b did not cross-react with heterologous IgG. Based on the absence of cross-reactions by IgG2a-specific monoclonal autoantibodies, certain peptides of the IgG CH2 and CH3 domains appear to generate the antigenic determinants of the anti-IgG2a RF in MRL/l mice. All of the monoclonal RF bound to Fc and, with one exception, not to Fab fragments of murine IgG. Binding of the monoclonal RF to substrate IgG was not inhibited by Clq, thus excluding the Clq-binding site at the CH2 domain as one of the responsible epitopes in the induction of MRL/l RF. mIgMRF could be categorized as strongly, weakly, or noninhibitable by protein A, which interacts with IgG molecules at or near the CH2-CH3 junction. Inhibition appears to be caused by conformational changes and/or steric shielding of certain IgG areas distant from this junction and not by identical binding sites between protein A and RF. Certain of the mIgMRF that were weakly or not at all inhibitable by protein A were found to cross-react equally well with human Fc (CH2-CH3 domains) and pFc' (CH3 domain) fragments, indicating that the binding site for these monoclonals is at the CH3 domain. Monoclonal RF were devoid of anti-double-strand DNA, anticollagen, or antipeptidoglycan pentapeptide cross-reactivity, but one of the monoclonals cross-reacted with histones, four with single-strand DNA, and one with both histones and single-strand DNA.