The ribosomal P proteins are necessary for GTPase activity during protein synthesis. In addition to antibodies to the P proteins, sera from lupus patients contain anti-rRNA activity. To determine whether lupus antiribosomal sera recognize the region of 28S rRNA recently proposed to form part of the ribosomal GTPase center, an rRNA fragment corresponding to nucleotides (nt) 1922-2020 was transcribed in vitro and tested for antigenicity. 18 of 24 (75%) lupus sera containing anti-P antibodies, but only 2 of 24 (8%) lupus sera without anti-P, immunoprecipitated this rRNA fragment (p less than 0.001). The binding was specific, since no significant differences were observed between anti-P positive and negative lupus sera in binding to the RNA fragment transcribed in the antisense orientation or to a control region of rRNA. The majority of sera tested protected a rRNA fragment of approximately 68 nucleotides. To evaluate the fine specificity of the anti-28S antibodies, deletions and site-directed mutations were made in the RNA fragment. The anti-28S antisera required nt 1944-1955 for recognition and were remarkably sensitive to destabilizing as well as nondestabilizing mutations in the stems of the RNA fragments. Detection of antiprotein and anti-RNA antibodies directed against a functionally related domain in the ribosome, together with the remarkable specificity of anti-28S antibodies, strongly suggests a direct role for this region of the ribosome in initiating and/or maintaining antiribosomal autoantibody production.