A multiplex polymerase chain reaction (mPCR) was used for simultaneous amplification of the staphylococcal nuc gene, encoding the thermostable nuclease (TNase), and the mecA gene, encoding the penicillin-binding protein 2a which is associated with staphyloccal methicillin resistance. A total of 219 staphylococcal strains were tested and the mPCR data were compared with coagulase production and in vitro oxacillin suscpetibility. The agreement was 100% for coagulase production and nuc amplification, and 97.7%, 96.8 and 97.3% for mecA amplication and oxacillin resistance tested with MIC determination, disk diffusion and agar screen methods, respectively. Discrepant results were due to non-S. aureus isolates with borderline MICs of oxacillin (1–8 μg/ml). In a pilot test the mPCR simultaneously amplified both genes of staphylococci in blood cultures. This mPCR is a rapid and reliable method for single-step identification of cultures of MRSA and may prove to be useful for direct application on clinical specimens.