Levels of the single-stranded DNA-binding protein Pur(alpha), previously implicated in control of both DNA replication and gene transcription, are altered during the CV-1 cell cycle. Just prior to the onset of S phase, Pur(alpha) levels drop precipitously, after which they recover nearly 8-fold throughout S and G2 to peak just after mitosis. As observed previously, Pur(alpha) binds the hypophosphorylated form of the retinoblastoma protein, Rb. Coimmunoprecipitation of Pur(alpha) and Rb reveals that the complex declines as cells enter S phase and does not reform as Pur(alpha) levels recover in S and G2. As Pur(alpha) levels recover, the protein is localized to nuclear foci containing newly replicated DNA, as determined by immunoelectron microscopy using different sized gold beads and antibodies against Pur(alpha) and bromodeoxyuridine-labeled DNA. These foci also contain cyclin A, and Pur(alpha) coimmunoprecipitates with cyclin A from extracts of cells in S and G2 phases. Pur(alpha) remains with these foci throughout G2, after the bulk of DNA synthesis has ceased. Changing levels of Pur(alpha) may affect Pur(alpha) functions at the onset of S phase and during progression to mitosis.