Enterovirus (EV) detection by a new commercially available reverse transcription (RT)-PCR assay (Penter RT-PCR test) was compared with EV isolation from cell cultures and with EV detection by an in-house RT-PCR assay. Of the 54 cerebrospinal fluid specimens collected during a summer outbreak of aseptic meningitis, 52% were positive by cell culture versus 76% by in-house RT-PCR assay and 80% by the new RT-PCR test (52 versus 76 versus 80%; P = 0.003). This new reliable EV RNA detection test is suitable for clinical diagnosis of EV-related meningitis and may improve the management of EV-related neurological syndromes.