The cyclic peptide antibiotics capreomycin and viomycin are generally effective against the bacterial pathogen Mycobacterium tuberculosis. However, recent virulent isolates have become resistant by inactivation of their tlyA gene. We show here that tlyA encodes a 2'-O-methyltransferase that modifies nucleotide C1409 in helix 44 of 16S rRNA and nucleotide C1920 in helix 69 of 23S rRNA. Loss of these previously unidentified rRNA methylations confers resistance to capreomycin and viomycin. Many bacterial genera including enterobacteria lack a tlyA gene and the ensuing methylations and are less susceptible than mycobacteria to capreomycin and viomycin. We show that expression of recombinant tlyA in Escherichia coli markedly increases susceptibility to these drugs. When the ribosomal subunits associate during translation, the two tlyA-encoded methylations are brought into close proximity at interbridge B2a. The location of these methylations indicates the binding site and inhibitory mechanism of capreomycin and viomycin at the ribosome subunit interface.