A new enzyme immunoassay (EIA) for direct detection of enteroviruses based on a group-specific monoclonal antibody was evaluated using stool samples from patients with suspected enteroviral infection. The EIA was compared with polymerase chain reaction (PCR) and virus isolation in cell culture. Of 204 samples tested, 20 were positive by EIA, 34 by PCR, and 18 by cell culture. Compared with PCR, the most sensitive method, the sensitivity of EIA was 58% (20/34); the sensitivity of cell culture isolation was 52% (18/34). The results of both assays correlated in only 60% of cases. The combination of EIA and cell culture isolation detected 76% of PCR-positive stool samples. Enterovirus EIA provides results within 3-4 hr and requires only standard EIA equipment. It represents a rapid, reliable, and cost-effective diagnostic tool for enterovirus diagnosis from faecal samples. Negative results must be confirmed by other techniques, such as PCR or virus isolation in cell culture. J. Med. Virol. 60:439-445, 2000. (C) 2000 Wiley-Liss, Inc.