The Saccharomyces cerevisiae CDC46 protein accumulates in the nucleus of nondividing interphase cells. Soon after the cell has become committed to division, there is a rapid disappearance of CDC46 protein from the nucleus. This shift is not a consequence of variation in total CDC46 protein levels and is associated with DNA replication at the G1-S boundary. We propose that the CDC46 protein, which is required for DNA replication, becomes mobilized quickly from the cytoplasm into the nucleus as mitosis is completed and persists there until the next round of division is initiated. This partitioning of proteins in a cell cycle-dependent manner illustrates a general means of regulating events that must occur only once in each cycle, such as DNA initiation.