Enzyme immunoassays (EIAs) can be used to detect rotavirus antigens accurately in human stool specimens. Conventional EIA systems require prolonged incubations and multiple washing steps, thus making them impractical for truly rapid diagnosis. However, more rapid solid-phase EIA systems can be devised which make use of simultaneous binding of enzyme-labeled and solid-phase antibodies at immunologically distinct antigenic sites. A multiple-determinant EIA system was devised that was capable of detecting small quantities of rotavirus antigen in less than 40 min. This assay system accurately detected rotavirus antigen in 45 stool specimens positive for rotavirus. No false-positive reactions were noted provided that appropriate control reactions were performed. The rapid double-determinant EIA system provides an accurate, objective means for the rapid diagnosis of human viral infections.