Modifying RNA enzymes are highly specific for substrate—rRNA or tRNA—and the target position. In Escherichia coli , there are very few multisite acting enzymes, and only one rRNA/tRNA dual-specificity enzyme, pseudouridine synthase RluA, has been identified to date. Among the tRNA-modifying enzymes, the methyltransferase responsible for the m 2 A synthesis at purine 37 in a tRNA set still remains unknown. m 2 A is also present at position 2503 in the peptidyl transferase center of 23S RNA, where it is introduced by RlmN, a radical S-adenosyl-L-methionine (SAM) enzyme. Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. The Δ rlmN mutant lacks m 2 A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. Moreover, RlmN performs m 2 A 37 synthesis in vitro using a tRNA chimera as a substrate. This chimera has also proved useful to characterize some tRNA identity determinants for RlmN and other tRNA modification enzymes. Our data suggest that RlmN works in a late step during tRNA maturation by recognizing a precise 3D structure of tRNA. RlmN inactivation increases the misreading of a UAG stop codon. Since loss of m 2 A 37 from tRNA is expected to produce a hyperaccurate phenotype, we believe that the error-prone phenotype exhibited by the Δ rlmN mutant is due to loss of m 2 A from 23S rRNA and, accordingly, that the m 2 A2503 modification plays a crucial role in the proofreading step occurring at the peptidyl transferase center.