In order to make a rapid and accurate diagnosis of respiratory syncytial virus (RSV) infection, nasal swabs obtained from 14 neonates suspected of having this disease were examined for the presence of RSV genome by reverse transcription (RT) and nested polymerase chain reaction (PCR) amplification, along with enzyme immunoassay (EIA), serum neutralization testing and virus isolation. The RT-PCR method was sensitive enough to detect a 0.1 50% tissue culture infectious dose (TCID50) per milliliter by nested PCR. The RSV antigen was detected from the samples at more than 100 TCID50 per milliliter by EIA. Nine patients were positive for the presence of RSV genome by first PCR on the day of admission, and eight were also positive by nested PCR even on the fifth hospital day. Among nine PCR positives, four patients were positive for EIA and five for virus isolation. No cases were serologically diagnosed. The cases that were negative for RT-PCR were also negative according to the other methods. In the clinical setting, the RT-PCR assay is more useful for diagnosis of RSV infection than other methods when the suspected cases are negative by EIA assay.