Herpes simplex virus type 2 (HSV-2) gene US3 encodes a serine-threonine protein kinase. We previously described the isolation of a US3-inactivated mutant which is able to replicate in Vero cells but not in murine macrophages. To learn more about the biological role of the US3 protein kinase, we have sought to identify the target proteins of the enzyme. Studies of in vitro phosphorylation with extracts of infected cells demonstrate that the US3 protein kinase is involved in phosphorylation of the UL12 alkaline nuclease in vitro, suggesting that the nuclease is a possible target of the protein kinase.