The cloning of murine tryptophanyl-tRNA synthetase revealed the existence of at least three messenger RNAs able to code for this enzyme. In most of the tissues tested, two major mRNA species were detected. They are produced by alternative polyadenylation and they share the same open reading frame. The deduced peptide sequence is highly homologous to bovine and human tryptophanyl-tRNA synthetases. In embryonic stem cells, a third type of mRNA was characterized. Surprisingly, this mRNA contains, at the C terminus of the open reading frame, a sequence coding for six additional amino acids. Southern blot and polymerase chain reaction analysis showed that the two open reading frames are encoded by the same gene. Thus, alternative splicing may generate two tryptophanyl-tRNA synthetase isoforms. This phenomenon is the first reported case for an aminoacyl-tRNA synthetase mRNA with two open reading frame isoforms. Moreover, to our knowledge, this is the first time that a peptide addition to the COOH terminus of a protein, by mRNA alternative splicing, is described. The extra hexapeptide, Cys-Phe-Cys-Phe-Asp-Thr COOH, resembles the consensus sequence found in C termini of Ras proteins.