Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m(6) (2)A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity-binding assays. We demonstrate that residues in alpha 4, alpha 5, and the alpha 5-alpha 6 linker are essential for methyltransferase function, including an aromatic residue on alpha 4 that likely forms stacking interactions with the substrate adenosine and basic residues in alpha 5 and the alpha 5-alpha 6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.