Cell surface alterations following herpes simplex virus infection were studied by scanning electron microscopy at different times after infection of chick embryo fibroblasts and Vero cells. Beginning at 4 h, an increasing number of cells showed numerous microvillus protrusions 0.12-0.18 micron in diameter and 0.3 micron in length. These structures could also be stained on Vero cells with fluorescent phalloidin, indicating the presence of filamentous actin within them. At 12 h, the number of chick embryo fibroblasts with virus-induced microvillus-like structures began to decrease. There was an increase in virus titer in the medium beginning at 24 h, and it was therefore considered unlikely that the microvilli induced earlier had any function in virus release. Similar to poxvirus-induced microvilli formation, DNA and protein synthesis inhibitors prevented the induction of microvilli, indicating the involvement of a late viral function. N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine inhibited the formation of herpesvirus-induced microvilli in chick embryo fibroblasts but not in Vero cells. As this inhibitor had no effect on virus replication in either cell type, it is likely that the inhibition of microvillus formation was due to an anticellular activity of the drug.