The herpes simplex virus (HSV) US3 gene encodes a serine/threonine protein kinase (PK). Although US3 PK is not essential for virus replication in cell culture, it plays an important role in the regulation of apoptosis in infected cells. However, the role of US3 PK in virus replication and pathogenicity is not well understood. The UL46 gene encodes virion tegument phosphoproteins, the properties and functions of which are poorly understood. In this study, it is shown that the UL46 protein of HSV type 2 (HSV-2) is affected strikingly by the presence of US3 PK. In the absence of US3 PK, UL46 protein was quite unstable, being much more susceptible to degradation. UL46 protein was undetectable in the extracellular virions of US3-deficient virus. Moreover, in vitro kinase assays using recombinant US3 PK show that UL46 protein is phosphorylated by the US3 PK, suggesting that UL46 can be a direct substrate for US3 PK in infected cells. Together, these findings shed new light on the physiological functions of US3 PK.