A 2-step 'semi-nested' enterovirus PCR was developed and applied to CSF and serum specimens from 27 consecutive patients with aseptic meningitis. CSF and sera from 8 patients with non-enteroviral diagnoses were included as negative clinical controls. Enterovirus RNA was detected in CSF by PCR in 15 of the patients with aseptic meningitis, compared with 6 by virus culture. Acute-phase sera proved positive for enterovirus RNA in 11 patients, thus increasing the number of PCR-positive patients to 18. Convalescent-phase sera were all negative by PCR. The correlation of a positive or negative PCR result in CSF and/or serum versus combined conventional virology (serology and isolation from 1-3 sites, i.e. CSF, stool and throat) was 78%. All negative controls were negative by PCR. PCR is a reliable and sensitive diagnostic tool for the detection of enteroviral infections. Both CSF and acute-phase serum should be considered for testing.