A highly sensitive RNA-immunoprecipitation assay (Lerner-Steitz assay) is a unique and useful method of identifying autoantibodies to RNA-associated antigens. In this study, we identified novel autoantibodies to tRNAs using RNA-immunoprecipitation assay. In screening of 1472 sera by RNA-immunoprecipitation using HeLa cell extracts as an antigen source, 41 sera were found to immunoprecipitate tRNAs. Fifteen of these 41 sera also immunoprecipitated deproteinized tRNAs, indicating that these 15 sera contained anti-tRNA antibodies. Three sera immunoprecipitated naked tRNA from E. coli. When in vitro transcripts from cDNAs encoding E. coli tRNAs and their synthesized mutants were used as antigens, it was demonstrated that the representative serum recognized the conformational epitope of the "L"-shape structure which was conserved in all tRNAs of all species. This finding suggests the role of bacterial infection in the development of autoantibodies and autoimmune diseases. Two of 15 sera containing anti-tRNA antibodies were identified as anti-PL-12 (alanyl-tRNA synthetase) antibodies. Eleven of the remaining 13 patients were diagnosed as either SLE, Sjögren's syndrome or their overlap. In addition, fever, Raynaud's phenomenon, polyarthritis, leukocytopenia and characteristic annular erythema were frequently found in these patients. Novel autoantibodies to tRNAs appeared to be associated with common clinical features but were distinct from previously described anti-aminoacyl-tRNA synthetases.