The HSV-1 thymidine kinase (TK) gene of Herpes simplex virus was inserted into plasmids pBR322, pMOB45 and pHA10. The recombinant hybrid plasmids were used to transfect a tdk- mutant of Escherichia coli (Ky895) and the synthesis of the viral TK in the bacterial host was studied. Recombinant plasmids containing the entire BamHI-BamHI DNA fragment carrying the viral TK gene and the upstream sequences containing its promoter were able to produce the thymidine kinase, but removal of the BamHI-BglII DNA fragment containing the viral promoter of the TK gene resulted in enhanced expression of the viral gene, originating from the pBR322 tetr gene promoter. When the BglII-BamHI DNA fragment containing the viral TK gene was cloned in a plasmid with temperature-dependent copy number control (pMOB45), expression of the TK gene was enhanced fourfold by the temperature shift. Cloning of the HSV-1 TK gene in plasmid pHA10 showed that the lambda pL promoter allowed transcription of the viral gene but not the synthesis of active TK. The BamHI-BglII DNA fragment containing the viral upstream sequences with the promoter of the TK gene was found to contain transcription-termination sequences that prevented expression of the lambda kil gene of pHA10 in the presence of lambda N function.