The herpes simplex virus ICP4 protein is required for induction of early and late viral gene transcription as well as for repression of expression of its own gene and several other viral genes. Several electrophoretic forms of ICP4 have been observed, and phosphorylation is thought to contribute to this heterogeneity and possibly to the multiple functions of ICP4. To define the complexity of the site(s) of phosphorylation of ICP4 and to initiate mapping of this site(s), we have performed two-dimensional phosphopeptide mapping of wild-type and mutant forms of ICP4 labeled in infected cells or in vitro. Wild-type ICP4 labeled in infected cells shows a complex pattern of phosphopeptides, and smaller mutant forms of ICP4 show progressively fewer phosphopeptides, arguing that multiple sites on ICP4 are phosphorylated. The serine-rich region of ICP4, residues 175 to 198, was shown to be a site for phosphorylation. Furthermore, the serine-rich region itself or the phosphorylation of this region increases phosphorylation of all phosphopeptides. A mutant ICP4 molecule lacking the serine-rich region showed low levels of phosphorylation by protein kinase A or protein kinase C in vitro. These results suggest that there may be a sequential phosphorylation of ICP4, with phosphorylation of the serine-rich region stimulating phosphorylation of the rest of the molecule. In addition, purified ICP4 showed an associated kinase activity or an autophosphorylation activity with properties different from those of protein kinase A or protein kinase C.