Herpes simplex virus type 2 (HSV-2) gene US3 has been shown to encode a serine-threonine protein kinase. In this study, we have tried to identify target proteins of the US3 protein kinase using a US3 lacZ insertion mutant of HSV-2. When permeabilized cells were labelled with [gamma-32P]ATP under the optimum conditions for the US3 enzyme, the most striking difference between wild-type HSV-2 strain 186- and mutant-infected cells was observed in the phosphorylation of proteins ranging in M(r) values from 14K to 22K. Studies of in vitro phosphorylation with purified virions and with cells infected with a US9-defective HSV-1 mutant suggested that a tegment phosphoprotein encoded by the US9 gene may be a target of HSV-2 US3 protein kinase.