Abstract: DNA replication initiation in S. cerevisiae is promoted by B-type cyclin dependent kinase (Cdk) activity. In addition, once-per-cell cycle replication is enforced by cyclin-Cdk-dependent phosphorylation of the pre-replicative complex (pre-RC) components, Mcm2-7, Cdc6 and Orc1-6. Several of these controls must be simultaneously blocked by mutation in order to obtain re-replication. We looked for but did not obtain strong evidence for cyclin specificity in the use of different mechanisms to control re-replication: both the S phase cyclin Clb5, and the mitotic cyclins Clb1-4, were inferred to be able to impose ORC-based and MCM-based controls. We found evidence that the S-phase cyclin Clb6 could promote initiation of replication without blocking re-initiation, and this activity was highly toxic when the ability of other cyclins to block reinitiation was prevented by mutation. The failure of Clb6 to regulate re-initiation was due to rapid Clb6 proteolysis, since this toxic activity of Clb6 was lost when Clb6 was stabilized by mutation. Clb6-dependent toxicity is also relieved when early accumulation of mitotic cyclins is allowed to impose re-replication controls. Cell-cycle timing of re-replication control is crucial: sufficient re-replication block activity must be available as soon as firing begins. DNA re-replication induces DNA damage, and when re-replication controls are compromised, the DNA damage checkpoint factors Mre11 and Rad17 provide additional mechanisms that maintain viability and also prevent further re-replication, and this probably contributes to genome stability.