The presence of the ileS-2 gene, responsible for mupirocin resistance, in clinical isolates of methicillin-resistant Staphylococcus aureus was determined by multiplex polymerase chain reaction. Three pairs of primers were used, which yielded specific fragments of femA (encoding a unique feature of S. aureus), mecA (encoding resistance to methicillin) and ileS-2 genes. The multiplex polymerase chain reaction system is an easy and time-saving technique that, together with a rapid method for DNA extraction by boiling, may be incorporated as a routine analysis in clinical diagnostic laboratories.