Eukaryotic replication origins are'licensed'for replication early in the cell cycle by loading Mcm (2–7) proteins. As chromatin replicates, Mcm (2–7) are removed, thus preventing the origin from firing again. Here we report the purification of the RLF-B component of the licensing system and show that it corresponds to Cdt1. RLF-B/Cdt1 was inhibited by geminin, a protein that is degraded during late mitosis. Immunodepletion of geminin from metaphase extracts allowed them to assemble licensed replication origins. Inhibition of CDKs in metaphase stimulated origin assembly only after the depletion of geminin. These experiments suggest that geminin-mediated inhibition of RLF-B/Cdt1 is essential for repressing origin assembly late in the cell cycle of higher eukaryotes.