The ordered activation of the ubiquitin protein ligase anaphase-promoting complex (APC) or cyclosome by CDC20 in metaphase and by CDH1 in telophase is essential for anaphase and for exit from mitosis, respectively. Here, we show that CDC20 can only bind to and activate the mitotically phosphorylated form of theXenopus and the human APC in vitro. In contrast, the analysis of phosphorylated and nonphosphorylated forms of CDC20 suggests that CDC20 phosphorylation is neither sufficient nor required for APC activation. On the basis of these results and the observation that APC phosphorylation correlates with APC activation in vivo, we propose that mitotic APC phosphorylation is an important mechanism that controls the proper timing of APCCDC20activation. We further show that CDH1 is phosphorylated in vivo during S, G2, and M phase and that CDH1 levels fluctuate during the cell cycle. In vitro, phosphorylated CDH1 neither binds to nor activates the APC as efficiently as does nonphosphorylated CDH1. Nonphosphorylatable CDH1 mutants constitutively activate APC in vitro and in vivo, whereas mutants mimicking the phosphorylated form of CDH1 are constitutively inactive. These results suggest that mitotic kinases have antagonistic roles in regulating APCCDC20and APCCDH1; the phosphorylation of APC subunits is required to allow APC activation by CDC20, whereas the phosphorylation of CDH1 prevents activation of the APC by CDH1. These mechanisms can explain the temporal order of APC activation by CDC20 and CDH1 and may help to ensure that exit from mitosis is not initiated before anaphase has occurred.