Rotaviruses display in vivo a specific tropism for enterocytes of the small intestine. We examined here the infection of cultured human intestinal epithelial Caco-2 cells by rhesus monkey rotavirus (RRV) and human Wa rotavirus. The maximal infection of these cells was obtained when trypsin was present both in the viral inoculum before adsorption to the cells and in the culture medium during the course of cell infection. Since the differentiation process of Caco-2 cells in culture closely mimics in vivo differentiation of enterocytes along the crypt-villus axis, cell infection by RRV and Wa rotavirus was examined as a function of cell differentiation. We showed that RRV and Wa rotavirus can infect equally well both undifferentiated and differentiated Caco-2 cells.