Fission yeast replication checkpoint kinases Rad3p and Cds1p are essential for maintaining cell viability after transient treatment with hydroxyurea (HU), an agent that blocks DNA replication. Although current studies have focused on the cyclin-dependent protein kinase Cdc2p that is regulated by these checkpoint kinases, other aspects of their functions at the onset of S phase arrest have not been fully understood. In this study, we use genome-wide DNA microarray analyses to show that HU-induced change of expression profiles in synchronized G(2) cells occurs specifically at the onset of S phase arrest. Induction of many core environmental stress response genes and repression of ribosomal genes happen during S phase arrest. Significantly, peak expression level of the MluI-like cell cycle box (MCB)-cluster (G(1)) genes is maintained at the onset of S phase arrest in a Rad3p- and Cds1p-dependent manner. Expression level maintenance of the MCB-cluster is mediated through the accumulation of Rep2p, a putative transcriptional activator of the MBF complex. Conversely, the FKH-cluster (M) genes are repressed during the onset of S phase arrest in a Rad3p-dependent manner. Repression of the FKH-cluster genes is mediated through the decreased levels of one of the putative forkhead transcription factors, Sep1p, but not Fkh2p. Together, our results demonstrate that Rad3p and Cds1p modulate transcriptional response during the onset of S phase arrest.