A multiplex polymerase chain reaction (PCR) that can simultaneously detect eubacterial isolates and the methicillin-susceptibility of staphylococcal isolates from cerebrospinal and peritoneal fluid samples was compared to conventional microbiological methods. Using conventional methods, bacteria were isolated from 8% (29/350) of the cerebrospinal fluid samples and from 5% (3/60) of the peritoneal fluid samples. All isolates except two Staphylococcus epidermidis isolates were also detected using the multiplex PCR. Coagulase-negative staphylococci and Staphylococcus aureus were correctly identified using both methods. The multiplex PCR can rapidly and simultaneously detect eubacteria, and the methicillin susceptibility of staphylococci from samples containing > or = 10(2) cfu/ml of bacteria.