Background: A highly sensitive PCR assay for early and rapid detection of enteroviral (EV) RNA in CSF is necessary to investigate the role of EV in acute neurological illnesses. Objectives: To evaluate and compare two PCR protocols (Titan one-tube RT-PCR and random primed RT-PCR) for detection of enteroviral RNA in CSF. Study design: The PCR protocols were evaluated for lower limit of input detection using log dilutions of five stock EV strains and an isolate of enterovirus-71 in minimum essential medium and three EV stock strains in CSF. The tests were also applied on 77 CSF samples, 46 from patients with suspected acute EV neurological illness and 31 from 'disease controls'. Results: Even though in the initial virus titration assays there was no statistically significant difference in the limit of input detection by Titan system and the random primed two-step PCR, the latter had a higher positivity rate when used on CSF samples from patients (20/46 vs. 10/46, P <0.01). Conclusions: Random primed RT-PCR assay is superior to Titan one-tube RT-PCR for detection of EV RNA in CSF. (C) 2002 Elsevier Science B.V. All rights reserved.