We evaluated a new commercial enzyme immunoassay (EIA) for rotavirus (Rotavirus EIA; International Diagnostic Laboratories, Chesterfield, Mo). A total of 161 consecutive stool samples (including 18 from infants less than 30 days old) submitted to the diagnostic laboratory at Children's Hospital, Washington University Medical Center, St. Louis, Mo., for rotavirus detection were tested by Rotavirus EIA and by Rotazyme II (Abbott Laboratories, North Chicago, Ill.) according to the instructions of the manufacturer. In addition, 16 samples from infants less than 30 days old without diarrhea were tested by both assays. Samples showing discrepant results after repeat testing were examined by electron microscopy. Nine samples yielding discrepant results were also tested by using a reference EIA directly on the specimen and on culture supernatants from two passages in MA 104 cells. Rotavirus EIA and Rotazyme II yielded concordant results for 85% of the samples. All of the 26 discrepant samples tested negative by Rotavirus EIA and positive (15 samples) or equivocal (11 samples) by Rotazyme II. These samples included 11 from symptomatic infants more than 30 days old, 2 from symptomatic infants less than 30 days old (neonates), and 2 from neonates without diarrhea. Rotavirus was not detected in any of the 24 that were examined by electron microscopy or in any of the 9 that were tested by the reference EIA. The sensitivity, specificity, positive predictive value, and negative predictive value were 100% for Rotazyme EIA and 100, 90, 70, and 100%, respectively, for Rotazyme II. Rotavirus EIA was comparable to Rotazyme II in ease of performance. We conclude that Rotavirus EIA is equally sensitive and more specific than Rotazyme II for detecting rotavirus. Rotavirus EIA is a practical and accurate rotavirus assay for use in clinical laboratories.