Ionizing radiation potentiates the oncolytic activity of attenuated herpes simplex viruses in tumors exposed to irradiation at specific time intervals by inducing higher virus yields. Cell culture studies have shown that an attenuated virus lacking the viral gamma(1)34.5 genes underproduces late proteins whose synthesis depends on sustained synthesis of viral DNA. Here we report that ionizing radiation enhances gene expression from late viral promoters in transduced cells in the absence of other viral gene products. Consistent with this result, we show that in tumors infected with the attenuated virus, ionizing radiation increases 13.6-fold above baseline the gene expression from a late viral promoter as early as 2 hours after virus infection, an interval too short to account for viral DNA synthesis. The radiation-dependent up-regulation of late viral genes is mediated by the p38 pathway, inasmuch as the enhancement is abolished by p38 inhibitors or a p38 dominant-negative construct. The p38 pathway is not essential for wild-type virus gene expression. The results suggest that ionizing radiation up-regulates late promoters active in the course of viral DNA synthesis and provide a rationale for use of radiation to up-regulate cytotoxic genes introduced into tumor cells by viral vectors for cytoreductive therapy.