A simplified radioimmunoassay (RIA) technique was developed to facilitate the diagnosis of human rotavirus in stools of infants with diarrhea. This microtiter solid-phase RIA utilizes as a critical reagent hyperimmune serum against a tissue culture-grown simian rotavirus that is immunologically closely related to the human agent. One hundred thirteen pediatric stool samples were tested. Thirty-five specimens known to be positive for rotavirus by electron microscopy were also positive by RIA. All RIA-positive reactions (36) were shown to be specific for rotavirus by means of an antibody-blocking test. Nonspecific RIA-positive reactions were not encountered. This accurate, sensitive RIA technique is practical for use in diagnostic laboratories because critical serum reagents are prepared against culture-grown virus and not against rotavirus derived from carefully selected stools of humans or experimentally infected gnotobiotic animals.