We reported previously that the posttranslational processing associated with phosphorylation of the herpes simplex virus 1 infected-cell protein 22 (ICP22), a regulatory protein, is encoded by UL13, a gene encoding a structural protein of the virion. We now report the following. (i) In cells infected with a mutant lacking UL13 (delta UL13), restricted infected cells accumulate reduced levels of the regulatory protein ICP0 and several late viral proteins. Identical reductions have been observed in the same cell lines infected with a mutant from which the alpha 22 gene, encoding ICP22, had been deleted (delta alpha 22). We conclude that the UL13-mediated processing of ICP22 is essential for its gene-regulatory function. (ii) The reduced accumulations of specific viral protein in cells infected with either delta UL13 or delta alpha 22 viruses correlate with reduced levels of specific mRNAs for both ICP0 and the affected late genes. (iii) ICP22 is not modified by the UL13 protein introduced into cells during infection. (iv) ICP22 is also modified by the protein kinase encoded by US3, but this modification is different from that of the UL13 protein kinase. These results predict that UL13 encodes a protein kinase or phosphotransferase which is expressed late in the replicative life cycle and which directly or indirectly phosphorylates ICP22. This modification is essential for stabilization or increased transcription of a specific subset of viral RNAs and, ultimately, for the accumulation of corresponding viral proteins.