Enteroviruses are the most common pathogens identified in infants hospitalized for suspected aseptic meningitis. Rapid detection of enterovirus infection is essential in taking the decision for treatment with antiviral agents and applying infection control measures in hospitalized pediatric patients. The purpose of this study was to compare the results of conventional virus isolation with those of enteroviral RNA detection by reverse transcription (RT)-PCR method in identical specimens from cases of suspected aseptic meningitis. Cerebrospinal fluid (CSF) samples were collected for viral examination from 68 pediatric patients with suspected aseptic meningitis from 1999 to 2002. These samples were inoculated in HeLa, Hep-2 and RD cell culture. The viral RNA was investigated by in-house RT-PCR method. The isolated viruses were typed by neutralization test. 36 of the 68 specimens were detected to be enterovirus positive by culture method, while 43 of them yielded positive results when RT-PCR method is used. Discrepancies occurred between the two methods in 15 specimens. While 11 specimens were positive by RT-PCR, these are found to be culture-negative. The isolated viruses were typed as Echovirus 30 (n: 30), Group B coxsackievirus (n: 5) and one isolate could not be typed by neutralization. Because of higher sensitivity and rapidity of RT-PCR, it is superior (p = 0.016) to virus culture of CSF for the diagnosis of enterovirus meningitis. Although the clinical usefulness of viral culture from CSF is limited, the final laboratory identification needs cultural techniques.