A human cytomegalovirus (HCMV)-induced polypeptide of 68,000 Da (p68) with protein kinase activity was identified using a monoclonal antibody (F6b) produced against HCMV-infected cell proteins. p68 was detected by immunoprecipitation from 3 to 120 hr after infection and was induced by several strains of human and not simian CMV. Protein kinase activity was associated almost exclusively with nuclear HCMV-induced p68. Enzyme activity with ATP and casein as phosphate donor and acceptor, respectively, exhibited an optimum pH between 6 and 6.5. It was Mg2+ dependent and cAMP independent. The KATPm of 45 microM at pH 6.5 indicated a relatively high affinity of p68 for the nucleotide. p68 also transferred phosphate to phosvitin and light chains of F6b, as well as autophosphorylating at threonine and serine residues.