An enzyme-linked immunosorbent assay (ELISA), based on monoclonal antibodies to the astrovirus group antigen, was designed for the detection of astroviruses in stools of patients with gastroenteritis. Compared to immune electron microscopy used as the standard test, the sensitivity of the astrovirus ELISA was 91% (31/34) and the specificity was 96% (54/56). All five of the known astrovirus serotypes could be detected in 16 samples on which serotyping was done. In tests on 155 stools containing other enteric viruses, including adenoviruses, rotaviruses, caliciviruses, Hawaii virus, Snow Mountain virus, and Norwalk virus (30, 20, 70, 24, 4, and 7 samples, respectively), only 3 were positive in the astrovirus ELISA. The combined specificity for all astrovirus immune electron microscopy-negative samples was 98% (206/211). The results demonstrate that the new ELISA provides a sensitive and specific means for the diagnosis of astrovirus gastroenteritis.