The rRNAs ofEscherichia colicontain four 2′-O-methylated nucleotides. Similar to other bacterial species and in contrast with Archaea and Eukaryota, theE. colirRNA modifications are catalysed by specific methyltransferases that find their nucleotide targets without being guided by small complementary RNAs. We show here that theygdEgene encodes the methyltransferase that catalyses 2′-O-methylation at nucleotide C2498 in the peptidyl transferase loop ofE. coli23S rRNA. Analyses of rRNAs using MALDI mass spectrometry showed that inactivation of theygdEgene leads to loss of methylation at nucleotide C2498. The loss ofygdEfunction causes a slight reduction in bacterial fitness. Methylation at C2498 was restored by complementing the knock-out strain with a recombinant copy ofygdE.The recombinant YgdE methyltransferase modifies C2498 in naked 23S rRNA, but not in assembled 50S subunits or ribosomes. Nucleotide C2498 is situated within a highly conserved and heavily modified rRNA sequence, and YgdE's activity is influenced by other modification enzymes that target this region. Phylogenetically, YgdE is placed in the cluster of orthologous groups COG2933 together withS-adenosylmethionine-dependent, Rossmann-fold methyltransferases such as the archaeal and eukaryotic RNA-guided fibrillarins. TheygdEgene has been redesignatedrlmMforrRNAlarge subunitmethyltransferaseM.