Bacillus subtilis, which belongs to Gram-positive eubacteria, has been predicted to have a minor isoleucine tRNA transcribed from the gene possessing the CAT anticodon, which corresponds to methionine. We isolated this tRNA and determined its sequence including modified nucleotides. Modified nucleotide analyses using TLC, UV, and FAB mass spectroscopy revealed that the first letter of the anticodon is modified to lysidine [4-amino-2-(N⁶-lysino)-l-β-D-ribofuranosyl pyrimidine]. As a result, this tRNA agrees with the minor one predicted from the DNA sequence and is thought to decode the isoleucine codon AUA.