Replication factor A (RF-A) is a multisubunit, cellular protein that functions with SV40 T antigen during the initiation stage of DNA replication at the SV40 origin. It also cooperates with other replication factors to stimulate the activity of both polymerases alpha and delta during chain elongation. RF-A from both human and yeast cells is phosphorylated in a cell-cycle-dependent manner; the protein is phosphorylated at the G1- to S-phase transition, and dephosphorylation occurs at mitosis, thereby resetting this cycle. This observation provides a direct link between a protein required for DNA replication and cell-cycle-regulated protein phosphorylation.