p25rum1 from the fission yeast S. pombe is shown to act as a specific in vitro inhibitor of the p34cdc2/p56cdc13 mitotic kinase. It is also shown that early G1 cells contain p25rum1, which associates with and inhibits the mitotic kinase, and maintains p56cdc13 mitotic B cyclin at a low level, ensuring that these cells do not undergo a premature lethal entry into mitosis. A high level of p25rum1 in G2 cells inhibits the p34cdc2/p56cdc13 kinase that removes the block preventing a further S phase and leads to repeated rounds of DNA replication. Thus, the cyclin-dependent kinase inhibitor p25rum1, acting on the p34cdc2 mitotic kinase, plays an important role in ensuring the correct sequence of S phase and mitosis during the cell cycle.