DNA replication in eukaryotic cells must be strictly regulated to ensure that the entire genome is duplicated only once in each cell cycle. For this purpose, the initiation of DNA replication is controlled by the "licensing" reaction, which is established by the formation of a pre-replicative complex (pre-RC) with the sequential assembly of the origin recognition complex (ORC), Cdc6, Cdt1 and Mcm2-7 onto origin regions. Among these, Cdt1 is likely the most important target for regulating licensing in higher eukaryotic cells, since illegitimate accumulation of Cdt1 causes multiple rounds of DNA replication without an intervening mitosis. Cdt1 is regulated over the course of the cell cycle mainly by the controlled expression of an inhibitor protein, geminin, and the level of Cdt1 periodically fluctuates due to ubiquitination and proteolysis. While the expression of geminin from S phase to metaphase of mitosis prevents licensing, Cdt1 accumulates from M to G1 phases and is degraded at the onset of S phase. Furthermore, Cdt1 is also proteolyzed in G1 phase in response to DNA damage, presumably providing a new checkpoint control.