Abstract: The Cdc28p cyclin-dependent kinase is thought to both catalyze the onset of DNA replication and prevent rereplication by blocking the reassembly of initiation complexes at replication origins. Budding yeast with mutations in the CDC16 gene represent an exception to this model, because they rereplicate DNA despite being in a G2-like arrest with continually elevated Cdc28p kinase activity. We show, in contradiction to Pichler et al. (1997), that the extra DNA that accumulates in cdc16 mutants is largely chromosomal, as we originally reported. Two-dimensional DNA electrophoresis shows that cdc16 mutants reinitiate DNA synthesis from normal chromosome replication origins, and density transfer experiments show that multiple chromosomal locations are affected. Rereplication from origins requires both Cdc6p and Cdc46/Mcm5p, initiation proteins that had been thought to be inactivated by the Cdc28p kinase. These results establish that CDC16 is required to prevent inappropriate firing of replication origins.