Binding of reovirus type 1 to dispersed villus cells from the small intestine was found to be specific for the basolateral membrane. Reovirus type 3 did not bind to any surface of small intestinal epithelial cells, but did bind to intra-epithelial lymphocytes. Using reovirus genetic reassortants, it was shown that the viral attachment polypeptide, encoded by the S1 genome segment of reovirus type 1 is essential for reovirus specific immunofluorescence to villus epithelial cells. In addition, the binding of reovirus type 1 to intestinal cells is saturable. Competition for the binding of 125I-labeled reovirus type 1 was demonstrated with unlabeled reovirus type 1 but not unlabeled reovirus type 3. This indicates that the intestinal epithelial receptor for reovirus is not shared by reovirus serotypes 1 and 3, and infection of intestinal epithelial cells by reovirus type 3 may be limited due to a failure of virus to bind.