Background: Mucin, a population of polymeric glycoproteins, constitutes the primary component of the mucus layer that overlies the gastrointestinal tract. These studies aimed to determine whether murine intestinal mucins inhibit rotavirus infection. Methods: Murine intestinal mucins were obtained by scraping segments of mouse intestine and purification via CsCl gradient centrifugation and sepharose 4B chromatography. Inhibition of infection was determined by quantitation of immunoperoxidase-stained cells after infection with mucin-rotavirus mixtures. Results: Crude and purified intestinal mucins from suckling and adult mice are potent inhibitors of replication of a simian rotavirus, rhesus rotavirus (RRV), but weak inhibitors of other rotaviruses. In all preparations, colonic mucins were more potent inhibitors of RRV than small intestinal mucins. Suckling mucins neutralized RRV more effectively than adult mucins. In a panel of rotavirus reassortants, susceptibility to mucin inhibition correlated with the ability to hemagglutinate human type O erythrocytes and with RRV gene 4. Murine intestinal mucin inhibited RRV binding to MA104 cells, suggesting inhibition of virus-cell attachment to be the mechanism for neutralization. Mercaptoethanol or neuraminidase inhibited mucins' anti-RRV activities, implying the functional importance of mucins' polymeric structure and sialic acid content. Conclusions: These findings suggest that intestinal mucins represent a barrier to certain rotavirus infections.