The release of measles virus was studied in the presence of cytochalasin B (CB), a drug that disrupts actin microfilaments. In the presence of CB, infected cells accumulated infectious virus while virus released from these cultures decreased drastically (up to 99% inhibition). Electron micrographs showed that viral buds were reduced and had an unusual distribution along the cell membrane in CB-treated cultures. CB inhibition of released virus occurred rapidly (within 30 min) and to a full extent even when the drug was added during the final 2 hr of a 48-hr replicative cycle. CB inhibition of cellular functions is reversible and, similarly, inhibition of virus release could be almost completely reversed within 30 min after the drug was removed. Since CB can also inhibit sugar transport and protein glycosylation, 2-deoxy-D-glucose (DG) was used to study the manifestations of glycosylation inhibition. DG inhibited virus production only when added during the first one-third of the replicative cycle and inhibited cell-associated and released virus to an equal extent. Cytochalasin D, which disrupts microfilaments without affecting protein glycosylation, caused an inhibition of virus release analogous to the inhibition caused by CB. Thus, alteration of microfilament structure alters the normal budding process of measles virions. This suggests that microfilaments may play a role in the release of budding virions.