We report a system for investigating promoters of eucaryotic cell and virus genes based on analyses of the regulation of herpes simplex virus 1 (HSV-1) thymidine kinases whose structural gene sequences have been fused to the promoter of the gene under study. In infected cells, the polypeptides specified by HSV-1 form at least three groups, alpha, beta and gamma, whose synthesis is coordinately regulated and sequentially ordered at the transcriptional level. To identify the DNA sequence responsible for the regulation of transcription of alpha genes, we fused the sequence encoding the 5' end of an alpha gene to the structural gene sequence of the thymidine kinase, a beta gene. The resultant recombinant DNA was inserted into the viral genome and was also used to convert Ltk- cells to tk+ phenotype. In cells infected with recombinant virus, the thymidine kinase gene was regulated and expressed as an alpha gene-that is, it was transcribed and processed in the absence of prior infected cell protein synthesis. Moreover, mRNA selected by hybridization to sequences encoding the thymidine kinase contains at its 5' terminus sequences homologous to the donor sequence encoding the t'terminus of the alpha mRNA. In converted tk+ cells, the fused thymidine kinase gene, like the wild-type gene, is stimulated by superinfection with the tk- virus. However, the stimulation is many times greater and is due to non-alpha-gene products, whereas in cells converted by the wild-type gene, the stimulation is by alpha gene products. We conclude that the alpha genes are identified for transcription by sequences at or near those encoding the 5' terminus of the mRNA, and transposition of these sequences to a beta gene is all that is required to convert it to an alpha gene. Transcription of alpha genes appears to be regulated by non-alpha-gene products, which could be contained within the structure of the virion. In converted Ltk+ cells, the thymidine kinase gene uses its own promoter.