The major target of human cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) is the tegument protein CMVpp65. However, this protein has protein kinase (PK) activity, and the unknown effects on cell replication of an exogenous PK in healthy cells could limit the use of CMVpp65 as a vaccine, especially in children. In this report we show that a point mutation converting lysine to asparagine at the invariant lysine (K436), an essential site for phosphotransfer, abolishes the threonine kinase activity. The mutant CMVpp65 maintains its immunologic target characteristics, including antibody and CTL reactivity. This kinase-deficient CMVpp65 is a candidate for evaluation in future CMV vaccine development.