A morphological change induced by syn– herpes simplex virus type 1 (HSV-1), polykaryocyte formation, was enhanced by treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) in A431 cells. TPA treatment decreased the number of stress fibers, but led to the development of spike-like filopodia and actin-containing long projections. Similar reorganization of actin filaments was observed in HSV-1-induced polykaryocytes. The actin filament-disrupting drug cytochalasin D, but not the microtubule-disrupting drug nocodazole, inhibited the effect of TPA on polykaryocyte formation, indicating that the actin microfilament system plays a key role in this event. HSV-1 glycoprotein D (gD) was present in the cytoplasm of HSV-1-infected cells and gD gene-transfected cells; its expression became prominent at long cell projections in the presence of TPA. These findings suggest that the reorganization of actin filaments and cell motility are associated with the enhancing effect of TPA on HSV-1-induced polykaryocyte formation.