Autoantibodies to aminoacyl-transfer RNA (tRNA) synthetases are common in the human autoimmune diseases polymyositis and dermatomyositis. Sera of the PL-12 specificity contain separate antibodies reacting with alanyl-tRNA synthetase and alanine tRNA (tRNAAla). The antibodies to tRNA recognize at least six distinguishable human tRNAAla species grouped into two sequence families. The antibody-reactive determinants on the tRNA were identified through ribonuclease protection and oligonucleotide binding experiments. The antibody binding site is a seven- to nine-nucleotide sequence containing the anticodon loop and requires an intact anticodon. No requirement for anticodon stem structure or sequence is observed, although the 5' portion of the stem is protected from nuclease attack. Antibodies from several patients appear to share the same specificitym, indicating that the antibodies are induced by a unique sequence feature in the immunogen.