Components of ORC (the origin recognition complex) are highly conserved among eukaryotes and are thought to play an essential role in the initiation of DNA replication. The level of the largest subunit of human ORC (ORC1) during the cell cycle was studied in several human cell lines with a specific antibody. In all cell lines, ORC1 levels oscillate: ORC1 starts to accumulate in mid-G1 phase, reaches a peak at the G1/S boundary, and decreases to a basal level in S phase. In contrast, the levels of other ORC subunits (ORCs 2-5) remain constant throughout the cell cycle. The oscillation of ORC1, or the ORC1 cycle, also occurs in cells expressing ORC1 ectopically from a constitutive promoter. Furthermore, the 26 S proteasome inhibitor MG132 blocks the decrease in ORC1, suggesting that the ORC1 cycle is mainly due to 26 S proteasome-dependent degradation. Arrest of the cell cycle in early S phase by hydroxyurea, aphidicolin, or thymidine treatment is associated with basal levels of ORC1, indicating that ORC1 proteolysis starts in early S phase and is independent of S phase progression. These observations indicate that the ORC1 cycle in human cells is highly linked with cell cycle progression, allowing the initiation of replication to be coordinated with the cell cycle and preventing origins from refiring.