Mutations in the cell-division-cycle genes CDC46 and CDC47 were originally isolated as suppressors of mutations in two other cell-division-cycle genes (CDC45 and CDC54). We found several combinations of mutations in these genes that result in allele-specific suppression and synthetic lethality, confirming that this set of genes forms a group of genetically interacting components. Here, we show that the other genes, like CDC46, are all involved in an early step of DNA replication, possibly initiation of DNA synthesis. Mutants defective in each of the four genes exhibit high rates of mitotic chromosome loss and recombination. The mutants appear also to accumulate chromosome damage that can be detected by a novel chromosome electrophoresis assay. Conditional mutants in this group, under fully nonpermissive conditions, show cell-cycle arrest at the beginning of DNA synthesis; under less stringent conditions, some arrest later, in S-phase. The DNA sequence of the CDC46 gene indicates that the protein is a member of a new family of genes apparently required for DNA initiation, with family members now identified in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mouse cells.