ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early protein that stimulates viral mRNA expression from many viral delayed-early and late genes during infection. One HSV-1 late gene which is highly dependent on ICP27 during infection is that encoding the glycoprotein C (gC). Here we report that the gC gene is specifically transactivated by ICP27 in transfected Vero cells. Using various gC plasmid constructs, we show that ICP27's stimulatory effects are independent of the gC gene's endogenous promoter and polyadenylation site. This suggests that ICP27-responsive elements lie in the transcribed body of the gC gene. We also show that transactivation of the gC gene by ICP27 is independent of other viral proteins, as ICP27 alone can transactivate the gC gene when its transcription is mediated by the human cytomegalovirus immediate-early gene promoter. However, when gC gene expression is driven by its endogenous promoter, the stimulatory effect of ICP27 requires additional transactivators. To explore the level at which ICP27 transactivates the gC gene, we established stably transfected Vero cell lines that have integrated copies of the gC gene under control of the cytomegalovirus immediate-early gene promoter. These gC genes are not constitutively expressed but can be efficiently induced by HSV-1 infection. Using nuclear run-on transcription assays, we show that transcriptional induction of the stably transfected genes is ICP27 independent. In contrast, accumulation of gC mRNA is very highly dependent on ICP27. Together, these results demonstrate that ICP27 posttranscriptionally activates mRNA expression from a biologically relevant viral target gene.