Proteolysis triggered by the anaphase-promoting complex (APC) is needed for sister chromatid separation and the exit from mitosis. APC is a ubiquitin ligase whose activity is tightly controlled during the cell cycle. To identify factors involved in the regulation of APC-mediated proteolysis, a Saccharomyces cerevisiae GAL-cDNA library was screened for genes whose overexpression prevented degradation of an APC target protein, the mitotic cyclin Clb2. Genes encoding G ₁ , S, and mitotic cyclins were identified, consistent with previous data showing that the cyclin-dependent kinase Cdk1 associated with different cyclins is a key factor for inhibiting APC ^Cdh1 activity from late-G ₁ phase until mitosis. In addition, the meiosis-specific protein kinase Ime2 was identified as a negative regulator of APC-mediated proteolysis. Ectopic expression of IME2 in G ₁ arrested cells inhibited the degradation of mitotic cyclins and of other APC substrates. IME2 expression resulted in the phosphorylation of Cdh1 in G ₁ cells, indicating that Ime2 and Cdk1 regulate APC ^Cdh1 in a similar manner. The expression of IME2 in cycling cells inhibited bud formation and caused cells to arrest in mitosis. We show further that Ime2 itself is an unstable protein whose proteolysis occurs independently of the APC and SCF (Skp1/Cdc53/F-box) ubiquitin ligases. Our findings suggest that Ime2 represents an unstable, meiosis-specific regulator of APC ^Cdh1 .