Nasal carriage of Staphylococcus aureus is considered a source of subsequent infection in health care settings. Utilization of real-time polymerase chain reaction (PCR) for detection of S. aureus has the potential to dramatically affect infection control practice by rapidly identifying S. aureus-colonized patients. We developed and validated the use of real-time PCR for detection of S. aureus colonization in two patient populations. Paired nasal swabs were collected from 299 neonates and from 151 adult patients at Evanston Hospital. One swab was used for culture and the other placed into a bacterial lysis solution containing achromopeptidase. The DNA liberated was used as the template for real-time PCR with primers for the femA gene. SYBR Green was used for amplicon detection. In the neonatal population the sensitivity, specificity, predictive value positive and predictive value negative for culture and PCR was 92% versus 96%, 100% versus 100%, 100% versus 100%, and 98% versus 99%, respectively. In the adults the results were 90% versus 100%, 100% versus 98%, 100% versus 96%, and 95% versus 100%, respectively. Real-time PCR was able to detect S. aureus in 2 hours compared to 1 to 4 days for culture and provided sensitivity equal to or greater than culture.