The intra-S cell cycle checkpoint is critical in controlling the cell cycle progression, halting the replication and allowing the time to repair to maintain genomic integrity when DNA damage and replication stresses occur. If this checkpoint becomes defective, cells lose their abilities to completely halt replication, repair the damage and result in replicative stress and genomic instability which will subsequently increase the risk of cancer. We have discovered that a large proportion of melanoma cell lines lose the ability to completely halt replication when challenged with high dose hydroxyurea-induced replicative stress, despite normal cell cycle checkpoint activation. Moreover, we found that failure in the crosstalk between cell cycle checkpoint activation, including activation of the checkpoint signalling kinase Chk1, and degradation of CDC25A, which is triggered by Chk1-dependent phosphorylation of CDC25A . CDC25A is a key regulator CYCLIN/CDK complexes that promote replication and cell-cycle progression through S phase. It is the uncoupling of checkpoint activation and CDC25A degradation that is likely to be accountable for this defective response to replicative stress in melanoma cells. Chk1 inhibitors selectively target cells with high levels of endogenous replicative stress, and this may provide an opportunity to selectively targets melanomas with this defective cell cycle response.