In eukaryotic cells a surveillance mechanism, the S phase checkpoint, detects and responds to DNA damage and replication stress, protecting DNA replication and arresting cell cycle progression. We show here that the S phase cyclins Clb5 and Clb6 are regulated in response to genotoxic stress in the budding yeast Saccharomyces cerevisiae. Clb5 and Clb6 are responsible for the activation of the specific Cdc28 cyclin-dependent kinase activity that drives the onset and progression of the S phase. Intriguingly, Clb5 and Clb6 are regulated by different mechanisms. Thus, the presence of Clb6, which is eliminated early in an unperturbed S phase, is stabilized when replication is compromised by replication stress or DNA damage. Such stabilization depends on the checkpoint kinases Mec1 and Rad53. The stabilization of Clb6 levels is a dynamic process that requires continued de novo protein synthesis, because the cyclin remains subject to degradation. It also requires the activity of the G1 transcription factor Mlu1 cell cycle box-binding factor (MBF) in the S phase, whereas Dun1, the checkpoint kinase characteristically responsible for the transcriptional response to genotoxic stress, is dispensable in this case. On the other hand, two subpopulations of endogenous Clb5 can be distinguished according to turnover in an unperturbed S phase. In the presence of replication stress, the unstable Clb5 pool is stabilized, and such stabilization requires neither MBF transcriptional activity nor de novo protein synthesis. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.