Early in eukaryotic cell cycle, a pre-RC is assembled at each replication origin with ORC, Cdc6, Cdt1 and Mcm2-7 proteins to license the origin for use in the subsequent S phase. Licensed origin must then be activated by S-Cdk and Ddk. At the onset of S phase, RPA is loaded on to the ARS in a reaction stimulated by S-Cdk and Ddk, followed by Cdc45-dependent loading of pol alpha, -delta, and -epsilon. This study examines cell cycle-dependent localization of pol alpha, -delta and -epsilon in Saccharomyces cerevisiae using immuno-histochemical and chromatin immuno-precipitation methods. The results show that pol alpha, -delta, or -epsilon localizes on chromatin as punctate foci at all stages of the cell cycle. However, some foci overlap with or are adjacent to foci pulse-labeled with bromodeoxyuridine during S phase, indicating these are replicating foci. DNA microarray analysis localized pol alpha, -delta, and -epsilon to early firing ARSs on yeast chromosome III and VI at the beginning of S phase. These data collectively suggest that bidirectional replication occurs at specific foci in yeast chromosomes and that pol alpha, -delta, and -epsilon localize and function together at multiple replication forks during S phase.