Internalization of herpes simplex virus type 1 (HSV) KOS strain by HEp-2 cells was reversibly inhibited by pretreatment of cells with cytochalasins B and D. Internalization of virus following preincubation at 4 degrees C and temperature shift to 37 degrees C was normally preceded by a 5 to 8 min lag period and was complete within 20 to 30 min. A similar lag period followed HSV addition at 37 degrees C. Cytochalasin D was fivefold more active on HSV entry than cytochalasin B, with 50% inhibition at 2 microM and 10 microM respectively. Inhibition was completely reversible, such that all cell-bound infectious virus was recovered upon removal of cytochalasin. In conjunction with previous reports, the activity of cytochalasin on HSV entry suggests that a change in cytoskeletal structure following virus attachment triggers a microfilament activity important for internalization of HSV by HEp-2 cells.