We reported here an efficient and generally applicable genomic analysis that uses transcriptional profiling to identify candidate substrates of regulatory enzymes, such as kinases and ubiquitin ligases. We applied this strategy to the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls sister chromatid separation and exit from mitosis. We found that a microtubule-associated protein, CKAP2, is a substrate of APC/C and demonstrated that ubiquitination and degradation of CKAP2 in vitro require a KEN-box and is mediated by Cdh1, an activator of APC/C. We showed that the levels of CKAP2 fluctuated across the cell cycle in culture cells, high in mitosis and low during mitotic exit. Overexpression of Cdh1 reduced the levels of CKAP2 in a KEN-box-dependent manner, while knockdown of Cdh1 increased the half-life of CKAP2. CKAP2 associated with centrosomal microtubules in late G(2), but only after the separation of the duplicated centrosomes. During mitosis, CKAP2 associated with spindle poles and with spindle microtubules from prophase through anaphase and dis-appeared from microtubules during cytokinesis. The function of CKAP2 during mitosis does not seem essential, as efficient knockdown of CKAP2 neither altered the cell cycle distribution of the cells, nor generated observable mitotic defects. On the other hand, ectopic expression of either the wild-type or a non-degradable CKAP2 led to a mitotic arrest with monopolar spindles containing highly bundled microtubules. We concluded that CKAP2 is a physiological substrate of APC/C during mitotic exit and that a tight regulation of the CKAP2 protein level is critical for the normal mitotic progression.