Ctf4p (chromosome transmission fidelity) has been reported to function in DNA metabolism and sister chromatid cohesion in Saccharomyces cerevisiae. In this study, a ctf4(S143F) mutant was isolated from a yeast genetic screen to identify replication-initiation proteins. The ctf4(S143F) mutant exhibits plasmid maintenance defects which can be suppressed by the addition of multiple origins to the plasmid, like other known replication-initiation mutants. We show that both ctf4(S143F) and ctf4Delta strains have defects in S phase entry and S phase progression at the restrictive temperature of 38 degrees C. Ctf4p localizes in the nucleus throughout the cell cycle but only starts to bind chromatin at the G1/S transition and then disassociates from chromatin after DNA replication. Furthermore, Ctf4p interacts with Mcm10p physically and genetically, and the chromatin association of Ctf4p depends on Mcm10p. Finally, deletion of CTF4 destabilizes Mcm10p and Pol alpha in both mcm10-1 and MCM10 cells. These data indicate that Ctf4p facilitates Mcm10p to promote the DNA replication.