Previous work has shown that a novel protein kinase is induced after infection of cultured cells with herpes simplex virus type 1 (HSV-1). Separately, it has been reported that the protein encoded by HSV-1 gene US3 shows similarity in its amino acid sequence to members of the protein kinase family of eukaryotes. We have investigated the possibility that these two observations are connected by preparing an antiserum to a synthetic oligopeptide corresponding to the carboxy-terminal eight amino acids of the US3 protein. This antiserum reacted on immunoblots with a polypeptide of apparent molecular weight 68,000 from extracts of cells which had been infected with HSV-1. The antiserum also reacted strongly with a 68,000 molecular weight species from a preparation of the novel HSV-1 protein kinase which had been extensively purified and resolved from other protein kinases. In addition, the purified preparation phosphorylated a protein species, also of 68,000 apparent molecular weight, when incubated with [gamma-32P]ATP. These data are consistent with gene US3 encoding the novel protein kinase induced after infection of cells with HSV-1.