Passage through the restriction point late in G1 normally commits cells to replicate their DNA. Here we show that the previously reported cell cycle block mediated by the human cytomegalovirus (HCMV) immediate early 2 (IE2) protein uncouples this association. First, IE2 expression leads to elevated levels of cyclin E-associated kinase activity via transcriptional activation of the cyclin E gene. This contributes to post-restriction point characteristics of IE2-expressing cells. Then these cells fail to undergo substantial DNA replication although they have entered S phase, and the induction of DNA replication observed after overexpression of cyclin E or D can be antagonized by IE2 without impinging on cyclin-associated kinase activities. These data suggest that IE2 secures restriction-point transition of cells before it stops them from replicating their genome. Our results fit well with HCMV physiology and support the view that IE2 is part of a viral activity which, on the one hand, promotes cell cycle-dependent expression of cellular replication factors but, on the other hand, disallows competitive cellular DNA synthesis.