Nick-translated DNA from a recombinant plasmid containing the herpes simplex virus type 1 thymidine kinase gene was used as a probe for the synthesis of thymidine kinase RNA. The recombinant plasmid was generated by inserting the 3.5-kilobase fragment derived by BamHI digestion of herpes simplex virus type 1 DNA into plasmid pBR322. At 8 h after infection, cytoplasmic and nuclear RNA hybridized to 14% and 19% of the recombinant DNA probe, respectively. However, no significant hybridization was found with either nuclear or cytoplasmic RNA extracted from cells infected and maintained in the presence of cycloheximide. This suggests that no thymidine kinase-related RNA was synthesized in the absence of alpha polypeptides, and supports the hypothesis that the alpha polypeptides effect new thymidine kinase RNA synthesis rather than being involved in processing or transport of thymidine kinase RNA. In cells infected and maintained in the presence of the arginine analog canavanine, about 2 to 3% of the plasmid DNA was found to hybridize with cytoplasmic and nuclear RNA. However, when a recombinant plasmid DNA containing only thymidine kinase coding sequences was used, no significant hybridization was found. The inhibition of thymidine kinase transcription by canavanine suggests that thymidine kinase belongs to the beta 2 kinetic class.