Proteins involved in the initiation of DNA replication play critical roles in the assembly and loading of replication complexes at replication origins. To gain further insight into the regulation of initiation, we screened in fission yeast for temperature-sensitive mutants which arrested at the G1/S boundary, and isolated nine mutants which arrested with a 1C DNA content at 36 degrees C. By linkage analysis, two complementation groups were identified which were not allelic to known G1 arrest mutations. One of the mutants isolated, sna41goul, arrested with a G1 DNA content and expressed a pleiomorphic phenotype, i.e., a mixture of cut and cdc phenotypes, at 36 degrees C. The point of arrest was identified as after START but before the hydroxyurea-induced block, by taking advantage of the mutant rad26.a14, which has a defect in an early S phase-specific checkpoint, and by performing reciprocal shift experiments. sna41 goal is allelic to sna41+, which is homologous to the CDC45 gene of budding yeast, and the mutation lies in a motif that is highly conserved in Cdc45-related proteins. The temperature sensitivity of the sna41goal mutant can be suppressed to some extent by ts mutations in polalpha. Our genetic results are consistent with a model in which Cdc45 plays crucial roles in the assembly of the replication apparatus at replication origins.