Herpes simplex virus 1 (HSV-1) UL13 is a viral protein kinase that is packaged into virions and regulates optimal expression of ICP0 and a subset of late (gamma) proteins, including UL41 in infected cells. In the present study, we investigated the role(s) of the protein kinase activity of UL13 in viral replication using a recombinant virus expressing enzymatically inactive UL13 after an amino acid substitution in the invariant lysine of UL13. The recombinant virus carrying this mutation formed smaller plaques yielded 10-fold less progeny than wild-type virus but could not be differentiated from wild-type virus with respect to accumulation of UL41 and ICP0 in infected cells. These results indicate that the protein kinase activity of UL13 plays a role in viral replication in cell culture, but the activity is not essential for the optimal expression of UL41 and ICP0.