A convenient rapid culture assay (RCA) for the detection of enteroviruses was evaluated against RT-PCR using 576 stool and 102 cerebrospinal fluid (CSF) samples. One hundred and ninety stool samples were also tested by conventional cell culture (CCC). The RCA used immunoperoxidase staining of cell culture plates with a blend of monoclonal antibodies (mAbs) against enterovirus VP1 on the second and sixth days after inoculation. This blend was composed of 5D8/1 (Dako) and four 'in-house' mAbs. CCC was performed using fluorescence staining with the Enterovirus Screening Set (Chemicon International) for culture confirmation. Detection of enteroviruses by the RCA was more successful in colonic carcinoma (CaCo-2) and rhabdomyosarcoma (RD) cells than in human embryonic lung fibroblasts, HEp2 and A549 cells. The performance of CCC in RD cells was hindered by rapid cell degeneration and non-specific staining of cells during culture confirmation. The sensitivity of the RCA compared to RT-PCR in stool samples was found to be 71 % (115/161) on the second day and 87 % (140/161) on the sixth day. The sensitivity of the RCA in CSF samples was 38 % (22/58) after 2 days and 59 % (34/58) after 6 days. The specificity of the RCA was 100 %. All CCC-positive samples were positive by the RCA. CCC required 3-14 days for virus recovery. In conclusion, the RCA has the same sensitivity as CCC, significantly shortens the time required for the detection of enteroviruses, and prevents pitfalls associated with using RD cells for CCC. For diagnosis of aseptic meningitis in CSF samples, RT-PCR should be performed.