The gene product of cfr from Staphylococcus sciuri confers resistance to chloramphenicol, florfenicol and clindamycin in Staphylococcus spp. and Escherichia coli. Cfr is not similar to any other known chloramphenicol resistance determinant. Comparative investigation of E. coli with and without a plasmid-encoded Cfr showed a decreased drug binding to ribosomes in the presence of Cfr. As chloramphenicol/florfenicol and clindamycin have partly overlapping drug binding sites on the ribosome, the most likely explanation is that Cfr modifies the RNA in the drug binding site. This hypothesis was supported by drug footprinting data that showed both a decreased drug binding and an enhanced reverse transcriptase stop at position 2504, which corresponds to a modification at position A2503 at the drug binding site. A 45 n long RNA fragment containing the appropriate region was isolated and MALDI-TOF mass spectrometry in combination with tandem mass spectrometry showed an additional methylation at position A2503. Moreover, reduced methylation was detected at nucleotide C2498. The results show that Cfr is an RNA methyltransferase that targets nucleotide A2503 and inhibits ribose methylation at nucleotide C2498, thereby causing resistance to chloramphenicol, florfenicol and clindamycin.