The initial site of virus replication during measles infection is in the epithelial cells of the respiratory tract. We have investigated measles virus infection of two types of polarized epithelial cells to determine if entry and/or release of the virus is confined to either the apical or the basolateral plasma membrane. The Caco-2 line of human intestinal epithelial cells and the polarized Vero C1008 monkey kidney cell line were grown on permeable supports and inoculated either through the apical or basolateral surfaces. Cells exposed to virus in the apical medium showed high levels of synthesis of virus-specific proteins, whereas no synthesis of viral proteins was detected in cells inoculated at the basolateral surface. Virus titers derived from apically infected cells were found to be about 1000-fold greater than titers derived from cells infected at the basolateral surface. Indirect immunofluorescence results also demonstrated that expression of measles viral antigens occurs at high levels only when input virions are inoculated at the apical surface. To investigate the localization of CD46 and moesin, which are receptors for measles virus, Caco-2 cells were incubated with monoclonal antibodies against CD46 or moesin followed by 125I-labeled anti-mouse Ig. The results indicate that CD46 is expressed preferentially on the apical membranes while moesin appears to be present at similar levels on both surfaces. Release of the virus was also examined and found to be polarized as well. Virus was released into the apical medium at up to 1000-fold higher titers than virus released into the basolateral medium. These results demonstrate that in two epithelial cell types measles virus preferentially enters and is released from epithelial cells in a polarized fashion through the apical plasma membrane.