Streptomyces ambofaciens produces the macrolide antibiotic spiramycin, an inhibitor of protein synthesis, and possesses multiple resistance mechanisms to the produced antibiotic. Several resistance determinants have been isolated from S. ambofaciens and studies with one of them, srmA, which hybridized with ermE (the erythromycin-resistance gene from Saccharopolyspora erythraea), are detailed here. The nucleotide sequence of srmA was determined and the mechanism by which its product confers resistance was characterized. The SrmA protein is a methyltransferase which introduces a single methyl group into A-2058 (Escherichia coli numbering scheme) in the large rRNA, thereby conferring an MLS (macrolide-lincosamide-streptogramin type B) type I resistance phenotype. A mutant of S. ambofaciens in which srmA was inactivated was viable and still produced spiramycin, indicating that srmA is dispensable, at least in the presence of the other resistance determinants.