The sequences of the anticodon of tRNAAla, tRNAThr, and tRNAVal from Bacillus subtilis W 168 were N-G-C, N-G-U, and N-A-C, respectively. A new minor constituent, N, occupied the first position of the anticodon of each tRNA. N was indentified as 5-methoxyuridine (mo5U, Figure 1) by comparison of its UV absorption spectra, Rf values in thin-layer chromatography using several solvent systems and mass spectra with those of chemically synthesized specimen.