A hybridoma secreting monoclonal antibodies against RNA polymerase I was produced by the fusion of myeloma cells with spleen cells from a nonimmunized MRL/lpr mouse which is known to produce autoantibodies to RNA polymerase I. The antibodies (McAb-2D11) belong to the IgG2b subclass, reacted specifically with the second largest (120 kDa) subunit of RNA polymerase I, and inhibited accurate transcription of cloned rat rDNA in a fractionated cell extract following immunoprecipitation of RNA polymerase I. McAb-2D11 did not inhibit RNA polymerase II-mediated transcription of the mouse metallothionein-I gene. Immunocytochemical procedures with biotinylated second antibody demonstrated specific immunolocalization of RNA polymerase I in the nucleus. These studies have (a) presented direct evidence that autoantibodies to functional RNA polymerase I are produced in a murine model of systemic lupus erythematosus, (b) demonstrated specificity of the monoclonal antibody for RNA polymerase I, and (c) provided a useful tool for the purification of RNA polymerase I and/or transcription factor(s) associated with RNA polymerase I.