S-phase onset is controlled, so that it occurs only once every cell cycle. DNA is licensed for replication after mitosis in G1, and passage through S-phase removes the license to replicate. In fission yeast, Cdc6/18 and Cdt1, two factors required for licensing, are central to ensuring once per cell cycle replication. We show that the human Cdt1 homologue (hCdt1), a nuclear protein, is present only during G1. After S phase onset, hCdt1 levels decrease, and it is hardly detected in cells in early S-phase or G2. hCdt1 can associate with the DNA replication inhibitor Geminin, however these two proteins are mostly expressed at different cell cycle stages. hCdt1 mRNA, in contrast to hCdt1 protein, is expressed in S-phase arrested cells and its levels do not change dramatically during a cell cycle, suggesting that proteolytic rather than transcriptional controls ensure the timely accumulation of hCdt1. Consistent with this view, proteasome inhibitors stabilize hCdt1 in S-phase. In contrast, hCdc6/18 levels are constant through most of the cell cycle and are only low for a brief period at the end of mitosis. These results suggest that the presence of active hCdt1 may be crucial for determining when licensing is legitimate in human cells.