The herpes simplex virus type 1 (HSV-1) ICP4 protein is a transcriptional activator of many eucaryotic RNA polymerase II promoters. The HSV-1 thymidine kinase gene (tk) promoter is induced by ICP4 and contains binding sites for the cellular transcription factors TFIID, Sp1, and CCAAT-binding proteins, each of which affects expression of the tk gene. In this study, the effects of mutations in these sites on the transcription of tk in the presence and absence of ICP4 were determined during viral infection. Only the TATA box was necessary for efficient expression in the presence of ICP4; however, ICP4 apparently can still induce tk transcription even when the TATA box is disrupted. Alteration of the Sp1 sites had a minor effect on ICP4-induced expression in comparison to a large effect in the absence of ICP4, indicating that ICP4 can operationally substitute for the function of the transcription factor Sp1. In addition, tk was still expressed with the kinetics of an early gene in the absence of binding sites for Sp1 and CCAAT-binding proteins.