PCR for the diagnosis of enterovirus infections is resource intensive but is increasingly used due to wide availability. Enzyme-linked immunosorbent assays (ELISAs) that detect heterotypical antibodies against enterovirus immunoglobulin M (IgM), IgA, and IgG were compared with reverse transcription-PCR by using primers specific to the 5' untranslated regions of 60 enterovirus species. The ELISAs were less sensitive than the PCR, and only the ELISA for IgM was highly specific. When retrospective diagnosis is important or when specimens are unsuitable for PCR, the ELISA has a limited role if PCR is not available.