Flavopiridol, roscovitine, and other inhibitors of Cyclin-Dependent Kinases (CDK) inhibit the replication of a variety of viruses in vitro while proving nontoxic in human clinical trials of their effects against cancer. Consequently, these and other Pharmacological CDK inhibitors (PCIs) have been proposed as potential antivirals. Flavopiridol potently inhibits all tested CDKs and inhibits the transcription of most cellular and viral genes. In contrast, roscovitine and other purine PCIs inhibit with high potency only CDK1, CDK2, CDK5, and CDK7, and they specifically inhibit the expression of viral but not cellular genes. The levels at which purine PCIs inhibit gene expression are unknown, as are the factors which determine their specificity for expression of viral but not cellular genes. We show herein that roscovitine prevents the initiation of transcription of herpes simplex virus type 1 (HSV-1) genes but has no effect on transcription elongation. We further show that roscovitine does not inhibit the initiation or elongation of cellular transcription and that its inhibitory effects are specific for promoters in HSV-1 genomes. Therefore, we have identified a novel biological activity for PCIs, i.e., their ability to prevent the initiation of transcription. We have also identified genome location as one of the factors that determine whether the transcription of a given gene is inhibited by roscovitine. The activities of roscovitine on viral transcription resemble one of the antiherpesvirus activities of alpha interferon and could be used as a model for the development of novel antivirals. The genome-specific effects of roscovitine may also be important for its development against virus-induced cancers.