We reconstructed the regulated induction of delayed-early (DE) transcription that occurs during herpes simplex virus (HSV) infection by using a transient expression system in which recombinant target genes were cotransfected into Vero cells together with intact activating genes. Plasmids containing cloned HSV-1 or HSV-2 immediate-early (IE) genes stimulated by up to 100-fold the expression from recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the DE promoter/regulatory region from the genes for an HSV-2 38,000-molecular-weight (38K) protein and the HSV-1 thymidine kinase. This activation was specific to hybrid genes containing DE regulatory regions since no significant increases in expression were observed in cotransfection experiments with the CAT gene without any promoter region or under the control of a number of other regulatory regions, including an HSV-1 IE regulatory region, the complete or enhancerless early regulatory region of simian virus 40, and an inducible cellular promoter/regulatory region. By using a variety of cotransfected plasmids containing individual or different combinations of HSV-1 or HSV-2 IE genes, we show that of the five known IE genes, two, those coding for the 175K and 110K polypeptides, each possessed the ability to stimulate expression from both DE promoters. Cleavage of the input plasmids within the known coding regions for the 175K and 110K proteins abolished stimulation of DE/CAT gene expression, whereas cleavage outside the coding regions had no effect on stimulation. We conclude that stimulation of CAT expression occurred exclusively by a transactivation mechanism in which the products encoded by these IE genes acted on the DE hybrid constructs at the transcription level. No transcriptional stimulatory function was demonstrated for the IE 68K and 63K proteins, although our results indicate that the IE 12K protein may augment the DE stimulatory activity of the 175K and 110K proteins.