Earlier studies have shown that the d120 mutant of herpes simplex virus 1, which lacks both copies of the alpha4 gene, induces caspase-3-dependent apoptosis in HEp-2 cells. Apoptosis was also induced by the alpha4 rescuant but was blocked by the complementation of rescuant with a DNA fragment encoding the U(S)3 protein kinase (R. Leopardi and B. Roizman, Proc. Natl. Acad. Sci. USA 93:9583-9587, 1996, and R. Leopardi, C. Van Sant, and B. Roizman, Proc. Natl. Acad. Sci. USA 94:7891-7896, 1997). To investigate its role in the apoptotic cascade, the U(S)3 open reading frame was cloned into a baculovirus (Bac-U(S)3) under the control of the human cytomegalovirus immediate-early promoter. We report the following. (i) Bac-U(S)3 blocks processing of procaspase-3 to active caspase. Procaspase-3 levels remained unaltered if superinfected with Bac-U(S)3 at 3 h after d120 mutant infection, but significant amounts of procaspase-3 remained in cells superinfected with Bac-Us3 at 9 h postinfection with d120 mutant. (ii) The U(S)3 protein kinase blocks the proapoptotic cascade upstream of mitochondrial involvement inasmuch as Bac-U(S)3 blocks release of cytochrome c in cells infected with the d120 mutant. (iii) Concurrent infection of HEp-2 cells with Bac-U(S)3 and the d120 mutant did not alter the pattern of accumulation or processing of ICP0, -22, or -27, and therefore U(S)3 does not appear to block apoptosis by targeting these proteins.