Previously, we reported that the U(S)3 protein kinase blocks apoptosis, that it activates protein kinase A (PKA), that activation of PKA blocks apoptosis in cells infected with a U(S)3 deletion mutant, and that an overlapping transcriptional unit encodes a truncated kinase designated U(S)3.5. Here, we report the properties of the kinases based on comparisons of herpes simplex virus and baculoviruses expressing U(S)3 or U(S)3.5 kinase. Specifically, we report the following. (i) Both kinases mediate the phosphorylation of HDAC1, HDAC2, and the PKA regulatory IIalpha subunit in the absence of other viral proteins. (ii) Both enzymes mediate the phosphorylation of largely identical sets of proteins carrying the phosphorylation consensus site of PKA, but only U(S)3 blocks apoptosis, suggesting that it is U(S)3 and not PKA that is responsible for the phosphorylation of the proteins bearing the shared consensus phosphorylation site and the antiapoptotic activity. (iii) Both kinases cofractionate with mitochondria. Immune depletion of the U(S)3 and U(S)3.5 kinases from the cytoplasm removed the kinases from the supernatant fraction, but not from the mitochondrial fraction, and therefore, if the antiapoptotic activity of the U(S)3 kinase is expressed in mitochondria, the localization signal and the antiapoptotic functions are located on different parts of the protein. (iv) The U(S)3 protein kinase is required for the translocation of virus particles from the nucleus. Although the U(L)31 protein is phosphorylated in cells infected with the mutant expressing U(S)3.5 kinase, the release of virus particles from nuclei was impeded in some cells, suggesting that the U(S)3 kinase affects the modification of the nuclear membrane more efficiently than the U(S)3.5 kinase.