Oncolytic viruses have been combined with standard cancer therapies to increase therapeutic efficacy. Given the sequential activation of herpes viral genes (herpes simplex virus-1, HSV-1) and the temporal cellular changes induced by ionizing radiation, we hypothesized an optimal temporal sequence existed in combining oncolytic HSV-1 with ionizing radiation. Murine U-87 glioma xenografts were injected with luciferase encoding HSV-1, and ionizing radiation (IR) was given at times before or after viral injection. HSV-1 replication and tumor-volume response were followed. Radiation given 6-9 h after HSV-1 injection resulted in maximal viral luciferase expression and infectious viral production in tumor xenografts. The greatest xenograft regression was also seen with radiation given 6 h after viral injection. We then tested if HSV-1 replication had a dose response to ionizing radiation. HSV-1 luciferase expression exhibited a dose response as xenografts were irradiated from 0 to 5 Gy. There was no difference in viral luciferase expression as IR dose increased from 5 Gy up to 20 Gy. These results suggest that the interaction of IR with the HSV-1 lytic cycle can be manipulated for therapeutic gain by delivering IR at a specific time within viral replicative cycle. © 2011 Macmillan Publishers Limited All rights reserved.