The requirement for the herpes simplex virus type 1 (HSV-1) protein Vmw65 (VP16) for activation of immediate early (IE) gene expression was examined in synchronized HeLa cells. Analyses of IE RNA levels were conducted during infection with a viral Vmw65 mutant, in1814. The results revealed an increased requirement for Vmw65 when cultures reached G2 phase of the cell cycle. The levels of IE RNAs 1, 2, and 4 were reduced 5-10 times more in G2 than G1/S for in1814-infected cells when compared to cells infected with wild-type virus or 1814R (a rescued virus), and similar but smaller effects were observed on IE RNA 3 levels. The relative decrease at G2 was reversed by resynchronization of cells to G1/S. Mutant in1814 formed plaques less efficiently on cells at G2 than on cells synchronized at G1/S. The results show that, in the absence of functional Vmw65, HSV-1 IE gene expression and replication vary during the cell cycle.