ABSTRACT Strains of methicillin-resistant Staphylococcus aureus (MRSA) have become the most important causative agents of hospital-acquired diseases worldwide. The genetic determinant of resistance, mecA , is not a gene native to S. aureus but was acquired from an extraspecies source by an unknown mechanism. We recently identified a close homologue of this gene in isolates of Staphylococcus sciuri , a taxonomically primitive staphylococcal species recovered most frequently from rodents and primitive mammals. In spite of the close sequence similarity between the mecA homologue of S. sciuri and the antibiotic resistance determinant mecA of S. aureus , S. sciuri strains were found to be uniformly susceptible to β-lactam antibiotics. In an attempt to activate the apparently “silent” mecA gene of S. sciuri , a methicillin-resistant derivative, K1M200 (for which the MIC of methicillin is 200 μg/ml), was obtained through stepwise exposure of the parental strain S. sciuri K1 (methicillin MIC of 4 μg/ml) to increasing concentrations of methicillin. DNA sequencing of the mecA homologue from K1M200 revealed the introduction of a point mutation into the −10 consensus of the promoter: the replacement of a thymine residue at nucleotide 1577 in the susceptible strain K1 by adenine in the resistant strain K1M200, which was accompanied by a drastic increase in transcription rate and the appearance of a new protein that reacted with monoclonal antibody prepared against the penicillin-binding protein 2A (PBP2A), i.e., the gene product of S. aureus mecA . Transduction of mecA from K1M200 (cloned into a plasmid vector) into a methicillin-susceptible S. aureus mutant resulted in a significant increase of methicillin resistance (from a methicillin MIC of 4 μg/ml to 12 and up to 50 μg/ml), the appearance of a low-affinity PBP detectable by the fluorographic assay, and the production of a protein that reacted in a Western blot with monoclonal antibody to PBP2A. Antibiotic resistance and the protein products disappeared upon removal of the plasmid-borne mecA homologue. The observations support the proposition that the mecA homologue ubiquitous in the antibiotic-susceptible animal species S. sciuri may be an evolutionary precursor of the methicillin resistance gene mecA of the pathogenic strains of MRSA.