Previous analyses using transient transfection assays indicated that the promoter for the gene encoding the herpes simplex virus type 1 (HSV-1) neurovirulence protein ICP34.5 can be divided into an essential core region of approximately 80 bp and two potent upstream silencer domains corresponding to the DR2 and DR6 repeat arrays. In order to examine the potential role of transcriptional silencing during productive HSV-1 infection, recombinant viruses were generated in which wild-type or mutant ICP34.5 promoters controlling the expression of a chloramphenicol acetyltransferase reporter gene were inserted into the thymidine kinase gene of the viral genome. The intact promoter in the virus HSV-delta1CAT exhibited delayed-early kinetics of expression that were comparable to those of the ICP34.5 gene promoter at its native site in the genome. Deletion of the core promoter domain eliminated promoter activity in the virus HSV-delta5CAT, indicating that this region was required for expression not only in transient transfections assays but also in the context of the viral genome. However, deletion of the DR2 repeat array from the ICP34.5 promoter in the virus HSV-delta7CAT was found to increase promoter activity only minimally at late times, and even to reduce activity at early times. Thus, in marked contrast to its behaviour in transient expression assays, the DR2 repeat array does not appear to act as a transcriptional silencer in the context of the HSV-1 genome during productive infection.