Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, we searched for high-copy suppressors of overproducer of Cdc25p (OPcdc25(+)), which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis (sum3(+)). This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p. We show that rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees and that cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees. These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA.