The initiation of DNA replication at replication origins in eukaryotic cells is tightly controlled to ensure that the genome is duplicated only once each cell cycle. We present evidence that in fission yeast, independent regulation of two essential components of the initiation complex, Cdc18 and Cdt1, contributes to the prevention of reinitiation of DNA replication. Cdc18 is negatively controlled by cyclin-dependent kinase (CDK) phosphorylation, but low level expression of a mutant form of Cdc18 lacking CDK phosphorylation sites (Cdc18(CDK)) is not sufficient to induce rereplication. Similar to Cdc18, Cdt1 is expressed periodically in the cell cycle, accumulating in the nucleus in G(1) and declining in G(2). When Cdt1 is expressed constitutively from an ectopic promoter, it accumulates in the nucleus throughout the cell cycle but does not promote reinitiation. However, constitutive expression of Cdt1, together with Cdc18(CDK), is sufficient to induce extra rounds of DNA replication in the absence of mitosis. Significantly greater levels of rereplication can be induced by coexpression of Cdc18(CDK) and a Cdt1 mutant lacking a conserved C-terminal motif. In contrast, uncontrolled DNA replication does not occur when either mutant protein is expressed in the absence of the other. Constitutive expression of wild-type or mutant Cdt1 also leads to an increase in the levels of Cdc18(CDK), possibly as a result of increased protein stability. Our data are consistent with the hypothesis that control of rereplication depends on a redundant mechanism in which negative regulation of Cdt1 functions in parallel with the negative regulation of Cdc18.