Different milk proteins were analyzed for their inhibitory effect on either rotavirus-mediated agglutination of human erythrocytes or rotavirus infection of the human enterocyte-like cell line HT-29. Proteins investigated were alpha-lactalbumin, beta-lactoglobulin, apo-lactoferrin, and Fe(3+)-lactoferrin, and their antiviral action was compared with the activity of mucin, a milk glycoprotein known to affect rotavirus infection. Results obtained demonstrated that beta-lactoglobulin, apo- and Fe(3+)-lactoferrin are able to inhibit the replication of rotavirus in a dose-dependent manner, apo-lactoferrin being the most active. It was shown that apo-lactoferrin hinders virus attachment to cell receptors since it is able to bind the viral particles and to prevent both rotavirus haemagglutination and viral binding to susceptible cells. Moreover, this protein markedly inhibited rotavirus antigen synthesis and yield in HT-29 cells when added during the viral adsorption step or when it was present in the first hours of infection, suggesting that this protein interferes with the early phases of rotavirus infection.