Expression of the early and late genes of herpes simplex virus type 1 (HSV-1) during infection of tissue culture cells requires the prior expression of the immediate early (IE) genes. The requirement for the product of IE gene 3, Vmw175, for the activation of early promoters has been revealed by studies with temperature-sensitive virus mutants. Recent experiments using transfection assays have shown that both Vmw175 and the product of IE gene 1, Vmw110, are involved in the transactivation of a variety of HSV-1 early promoters. This paper describes experiments which compared the activation of two early promoters [those of the glycoprotein gD and thymidine kinase (tk) genes] with that of a member of a later class of genes (the major capsid protein, VP5). Plasmids containing these promoters linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into HeLa cells with plasmids containing one or more HSV-1 IE genes. Promoter activity was estimated by measurement of CAT activity in extracts of transfected cells. The gD and tk promoters were activated by both Vmw175 and Vmw110, and the combination of these two IE gene products resulted in very high levels of activation. Addition of further IE gene products did not result in any significant increase in the activation seen with the combination of Vmw175 and Vmw110. In contrast, the activation of the VP5 promoter brought about by the combination of Vmw175 and Vmw110 was relatively slight, but was increased further when plasmids containing IE gene 2, encoding Vmw63, were included in the transfection. These data suggest that Vmw63, like Vmw175 and Vmw110, is also involved in the activation of transcription from HSV-1 promoters. The effect of Vmw63 may be limited to the activation of a subset of HSV-1 genes.