Abstract: We have isolated new mutants of the yeast Saccharomyces cerevisiae that are defective in mitotic DNA synthesis. This was accomplished by directly screening 11000 newly isolated temperature-sensitive yeast clones for DNA synthesis defects. Ninety-seven different mutant strains were identified. Approximately half had the fast-stop DNA synthesis phenotype; synthesis ceased quickly after shifting an asynchronous population of cells to the restrictive temperature. The other half had an intermediate-rate phenotype; synthesis continued at a reduced rate for at least 3 h at the restrictive temperature. All of the DNA synthesis mutants continued protein synthesis at the restrictive temperature. Genetic complementation analysis of temperature-sensitive segregants of these strains defined 60 apparently new complementation groups. Thirty-five of these were associated with the fast-stop phenotype, 25 with the intermediate-rate phenotype. The fast-stop groups are likely to include many genes whose products play direct roles in mitotic S phase DNA synthesis. Some of the intermediate-rate groups may be associated with S phase as well. This mutant collection should be very useful in the identification and isolation of gene products necessary for yeast DNA synthesis, in the isolation of the genes themselves, and in further analysis of the DNA replication process in vivo.FAU - Dumas, L .