||Eukaryotic DNA replication has been most extensively studied in the budding yeast Saccharomyces cerevisiae, because it has a small genome, powerful genetics and the ability of plasmid maintenance to provide a replication assay. While the DNA elements and many proteins involved in the initiation of DNA replication have been identified, some replication-initiation proteins may not have been discovered yet. We have developed a phenotypic screen to identify previously unknown proteins for the i̲nitiation of D̲NA r̲eplication (the “IDR” screen). A primary screen is first used to identify plasmid-loss candidates with defects in the initiation of DNA replication and/or plasmid segregation. In the secondary screen, a pair of plasmids, p1ARS and p8ARSs, is then used to distinguish idr mutants from other mutants, such as those in plasmid segregation. Furthermore, a tertiary screen is performed to exclude mutants in previously known replication-initiation proteins. A colony color system is employed for detecting plasmid loss in the primary, secondary and tertiary screens. We have screened about 380,000 EMS mutagenized colonies. Up to now, 29 mutants were found to be in known replication-initiation genes, while the plasmid loss phenotypes of 3 strains could not be complemented by the known genes. These 3 mutants are likely to contain mutations in previously unknown replication-initiation genes. We have cloned one of the idr mutants and are in the process of trying to identify the other two idr mutants by complementation of the plasmid loss phenotypes of the mutants with a yeast genomic library. CDC60 has been identified that can rescue both the plasmid loss and temperature-sensitivity (ts) phenotypes of an idr candidate C917. We found that HA- tagged Cdc60p associates with chromatin throughout the cell cycle. ChIP assay indicates that Cdc60p-HA can associate with ARS1 element. Cdc60p-HA can form a complex with Mcm2p, Mcm3p and Orc3p as shown by co-IP assay. On the other hand, two proteins Ipi3p and Rrp12p, identified from a collection of td (temperature-inducible degron) mutants, show typical plasmid loss phenotypes for individual mutants. Further experiments, including co-IP, cell cycle progression chromatin binding assay and 7-fractions chromatin binding assay illustrate that this two proteins associate with known initiation proteins and chromatin tightly. Ipi3p binds to chromatin in a cell cycle-regulated manner and Rrp12p binds onto chromatin throughout the cell cycle. Further characterization is still underway to unveil the mechanisms of Cdc60p, Ipi3p and Rrp12p in initiation of DNA replication.