AbstractDetection of human and bovine rotavirus in stools is described using a double‐antibody sandwich enzyme‐linked immunosorbent assay (ELISA) with polystyrene microtest plates as solid phase, immunoglobulin fraction of rabbit antiserum to rotavirus (human) as catching antibody, and the same reagent labelled with horseradish peroxidase as conjugate. The ELISA has been optimized with regard to simplicity, rapidity, sensitivity, and specificity. In a comparative study, stool specimens from 81 infants and children and 92 neonatal calves with diarrhoea were tested for rotavirus by ELISA, electron microscopy (EM), immunoelectro‐osmorphoresis (IEOP), and fluorescent antibody technique (FA). The relative sensitivity of the different assays for human and bovine rotavirus was: EM 68%, 76%; IEOP 80%, 76%; FA not determined, 85%; and ELISA 86%, 98%, respectively. Less than 1 ng of purified human rotavirus could be detected in ELISA, whereas 100 ng was the minimal amount detected by IEOP. It is concluded that the developed ELISA is a simple, rapid, reliable, and sensitive method for the diagnosis of human and bovine rotavirus infec