Conventional microbiology (CM) and real-time polymerase chain reaction (PCR) were used to determine rate and serotype of pneumococcal nasopharyngeal colonization in healthy children and children with upper respiratory illnesses (URI). One hundred and thirty-six healthy children and 79 children with URI were evaluated. Pneumococcal colonization was detected more often by real-time PCR than CM in healthy children (50% vs. 24%, p <or= 0.001), while detection rates were comparable by CM and real-time PCR in children with URI (61% vs. 65%, NS). Pneumococcal serotypes were identified 2.3 times more often in healthy children by real-time PCR than CM, p <or= 0.001 and 1.5 times more often in children with URI by PCR than CM, p = 0.01. Real-time PCR was also more sensitive in detecting multiple strains rather than CM in both healthy (p = 0.001) and children with URI (p <or= 0.001). Overall real-time PCR proved superior to CM in detection and serotyping of Streptococcus pneumoniae. Future studies should incorporate real-time PCR technology along with CM to fully understand the epidemiology of colonization in health and illness.