The p34CDC28 protein from Saccharomyces cerevisiae is a homolog of the p34cdc2 protein kinase, a fundamental regulator of cell division in all eukaryotic cells. Once activated it initiates the visible events of mitosis (chromosome condensation, nuclear envelope breakdown, and spindle formation). The p34CDC28 protein also has a critical role in the initiation of DNA synthesis. The protein kinase activity is regulated by cycles of phosphorylation and dephosphorylation and by periodic association with cyclins. An endogenous 40-kilodalton protein (p40) originally identified as a substrate of the p34CDC28 protein kinase was purified. The p40 protein bound tightly to p34CDC28 and inhibited the activity of the kinase. The p40 protein may provide another mechanism to regulate p34CDC28 protein kinase activity.