The diagnosis of a wide range of different neurological syndromes was established by a reverse transcription multiplex PCR assay. The presence of enterovirus and herpesviruses was studied in cerebrospinal fluid samples collected prospectively from 200 patients hospitalized with neurological diseases suspected of viral infection. Positive PCR results for enterovirus and neurotropic herpesvirus (herpes simplex, HSV, and varicella zoster, VZV) were obtained among the immunocompetent patients (55/156, 35%) who presented aseptic meningitis or encephalitis. Among immunocompromised patients the yield of positive PCR results was 41% (18/44), predominantly lymphotropic herpesviruses (15/44, 34%). Cytomegalovirus (CMV) DNA was detected in patients with several clinical syndromes, including encephalitis, chronic meningitis, retinitis, ventriculitis, polyradiculomyelitis, and myeloradiculitis. Epstein-Barr (EBV) and VZV-specific DNA sequences were detected in patients with either encephalitis, aseptic meningitis, and chronic meningitis. Dual infections of CMV and HSV or CMV and EBV were established in two AIDS patients with encephalitis and polyradiculomyelitis, respectively. The applications of this RT multiplex PCR assay are extensive and may prove to be particularly valuable for the rapid and sensitive diagnosis of neurological diseases in both immunocompetent and immunocompromised patients.