We previously reported that at least eight HSV-1 and five HSV-2 proteins were tyrosine phosphorylated in infected human and mouse cells and the first phosphotyrosine-modified gene product identified was the ICP22 regulatory protein (Blaho, J. A., Zong, C. S., and Mortimer, K. A., 1997, J. Virol. 71, 9828-9832). All electrophoretic forms of ICP22 are tyrosine phosphorylated with the exception of the fastest migrating (unmodified) isoform. We now report the following. (i) ICP22 that reacted with a specific anti-phosphotyrosine antibody contained a significant amount of phosphotyrosine based on phospho-amino acid analysis. These results validate the discovery of ICP22 tyrosine phosphorylation. (ii) Wild-type ICP22 extracted from infected HEp-2 cells migrated as at least seven isoforms, termed ICP22a-g, in denaturing gels. (iii) The primary structure of ICP22 possesses a sequence that is homologous to protein tyrosine kinase recognition sites. A virus, termed RF141, was generated in which ICP22 tyrosine(193) in the kinase target site was mutated to an alanine. (iv) Biochemical analyses of infected HEp-2 and primary HFF cells indicated that the distributions of ICP22 isoforms differed between RF141 and control HSV-1(F). (v) The accumulations of representative viral polypeptides in RF141-infected HEp-2 cells appeared similar to wild-type virus. (vi) RF141 had reduced efficiencies of plating in HFF cells compared to control Vero cells. These differences increased as the multiplicity of infection decreased. Based on these results, we conclude (vii) that ICP22 tyrosine(193) is required for optimal posttranslational modification of the protein in HSV-1 infected human epithelial HEp-2 and primary human fibroblast cells.