During herpes simplex virus type 1 (HSV-1) infection, the appearance of true-late gene products is severely reduced under conditions of DNA synthesis inhibition. This report describes the use of a plasmid-borne promoter of a true-late HSV-1 gene (US11), linked to the rabbit beta-globin gene, to study the requirement of DNA replication for late gene expression. The activity of the plasmid-borne US11 promoter in constructs containing or lacking an HSV-1 origin of replication (ORIS) was analysed by quantitative S1 mapping of correctly initiated hybrid transcripts. Following HSV-1 superinfection of transfected HeLa cells, the US11 promoter in ORI+ plasmids was expressed with similar kinetics to the viral US11 promoter. US11 promoter activity was first detected at the same time as the onset of DNA template replication. Expression of US11 RNA was detectable from non-replicating ORI- plasmids, although transcript accumulation was reduced by greater than 90%. Sequences containing the IE-5 promoter (a 3' co-terminal gene whose transcription starts 5' of US11) also played a positive role in achieving normal US11 gene expression.