A monoclonal antibody, 3F7, which reacts with the common rotavirus antigen on the sixth viral gene product was prepared. It was used in a direct monoclonal antibody radioimmunoassay (RIA) as a diagnostic reagent for detection in 3.5 hours of rotavirus in human pediatric stool specimens. In the 177 samples tested, a concordance of 96 percent was seen between the monoclonal RIA and the well-established and commonly used commercially available Rotazyme test. Six discrepant specimens which were positive by monoclonal RIA but negative by Rotazyme were shown to be positive by either electron microscopy or confirmatory blocking immunoassay. A seventh discrepant specimen was positive by Rotazyme and negative by monoclonal RIA as well as by both direct and immune electron microscopy. The monoclonal RIA test appears to be highly sensitive and specific, and merits additional evaluation as a rapid, convenient diagnostic assay that can reduce currently encountered problems associated with diagnosing rotavirus by immunoassay.