The replication factor Cdc45 has essential functions in the initiation and elongation steps of eukaryotic DNA replication and plays an important role in the intra-S-phase checkpoint. Its interactions with other replication proteins during the cell cycle and after intra-S-phase checkpoint activation are only partially characterized. Here, we present that the C terminal part of Cdc45 may mediate its interactions with Claspin. The interactions of human Cdc45 with the three replication factors Claspin, replication protein A (RPA) and DNA polymerase δ are maximal during S phase. Following UVC-induced DNA damage, Cdc45-Claspin complex formation is reduced whereas the binding of Cdc45 to RPA is not affected. We also show that treatment of cells with UCN-01 and Phosphatidylinisitol 3-kinase-like kinase inhibitors does not rescue the UV-induced destabilisation of Cdc45-Claspin interactions, suggesting that the loss of interaction between Cdc45 and Claspin occurs upstream of ATR activation in the intra-S-phase checkpoint. This article is protected by copyright. All rights reserved.