Rotavirus, the major viral enteropathogen causing acute gastroenteritis in children and young animals, infects the mature enterocytes of the villus epithelium of the small intestine. Relatively little is known about the molecular mechanisms involved in the rotavirus pathogenic process, and the cellular rotavirus receptor remains unknown. Infection of epithelial cells by some animal rotaviruses, but not human or most animal rotaviruses, requires the presence of sialic acid (SA) on the cell surface for efficient binding and infectivity. To further understand how rotaviruses enter susceptible cells to establish a productive infection, cultured African green monkey kidney (MAI04) and human colonic adenocarcinoma (HT-29 or Caco-2) epithelial cell lines grown on a permeable filter membrane supports containing 3.0 or 0.4 J.Lm pores were infected apically or basolaterally with SA-independent or SA-dependent rotaviruses. Caco-2 and HT-29, but not MAI04, cells developed polarized monolayers with well-developed brush borders and tight junctions. SA-dependent or SAindependent rotaviruses applied apically or basolaterally infected efficiently all cell lines grown on 3.0 J.Lm filter membranes. However, electron microscopy analysis revealed that these results were artefactual because all three cell lines penetrated through the 3.0 J.Lm pores and grew on the opposite face of the filter membranes. Cells grown on 0.4 J.Lm filter membranes did not penetrate through the pores. SA-independent rotaviruses applied apically or basolaterally were capable of efficiently infecting cells grown on 0.4 J.Lm filter membranes; SA-dependent rotaviruses only infected efficiently if applied apically. The presence of SA residues on either the apical or basolateral side was determined using a Texas Redconjugated lectin, wheat germ agglutinin (WGA), that binds SA residues. WGA bound exclusively to SA residues on the apical (Caco-2) or upper (MA104) surface of cells grown on 0.4 J.Lm filter membranes. These results confirmed the requirement of SA residues on the apical cell membrane for efficient infectivity of SA-dependent rotaviruses. These results also indicate that the rotavirus SA-independent cellular receptor is present on both sides of the epithelium. In addition, SA-dependent rotaviruses are less efficient than SA-independent rotaviruses at infecting the basolateral or lower surfaces of the cells analyzed, possibly due to the absence of SA residues on basolateral surfaces. Research supported by NIH DK30144.