Commercially available assay kits have now made detection of rotavirus in stool specimens possible as a routine laboratory test. One such kit, Rotazyme II (Abbott Laboratories, North Chicago, IL) has been reported to give a higher incidence of false positive results with neonatal stool than with stool from older patients. One hundred stool specimens from asymptomatic neonates (age range, two to five days) were tested by two ELISA methods and one latex agglutination method in order to evaluate the rate of false positivity in this group of patients. Negative staining electron microscopy was used as the reference method. The two ELISA methods were Rotazyme II and Rotavirus EIA (International Diagnostic Laboratories, St. Louis, MO), and the latex agglutination method was Meritec-Rotavirus (Meridian Diagnostics, Inc., Cincinnati, OH). The Rotavirus EIA and Meritec-Rotavirus tests gave 0% and 1% false positive results, respectively, while the Rotazyme II test gave a 4% false positive rate with an additional 19% equivocal results. This extensive comparative analysis of commercially available assays for detection of rotavirus in neonatal stool specimens suggests a false positive or equivocal rate with the Rotazyme II test that impairs clinical utility.