OBJECTIVE: To evaluate the Roche Amplicor polymerase chain reaction assay (APCR) by comparing the detection of enteroviruses from cerebrospinal fluid (CSF) by the Roche assay with detection by viral culture and to determine whether routine use of enteroviral PCR will affect patient management. METHODS: One hundred and sixty-three CSF specimens were tested by APCR and viral culture. Some of the discrepant specimens were resolved by retesting with an in-house PCR assay. Other discrepant results were resolved by testing the patients' serum by APCR or by viral culture of throat and stool specimens. RESULTS: Thirty CSF specimens were positive by APCR, and 18 of these were positive by viral culture. There were no APCR-negative, viral-culture-positive CSF specimens. Six of the 12 discrepant specimens were resolved as true positives. CONCLUSIONS: The APCR assay was more rapid and sensitive than viral culture for detection of enteroviruses from CSF. Routine use of this assay has the potential to reduce the amount of antibiotics used and the number of patient days spent in hospital.