Abstract A plethora of proteins and processes regulate proper mitosis and are yet to be fully understood. Since mitotic cells are an emerging target for cancer therapy, it is of utmost importance to decipher the mechanisms that regulate mitosis. The purpose of our study is to identify the role CUL3 plays in regulating mitosis. Specifically, it is our aim to identify CUL3 interacting partners that play roles in spindle assembly, in order to understand the function of CUL3 in constructing the mitotic spindle. The CUL3 protein is an ubiquitin E3 ligase scaffold. By means of ubiquitination, CUL3 can regulate an array of proteins. We have performed CUL3 RNAi experiments, followed by cell cycle analysis using flow cytometry and immunostaining of DNA, α-tubulin and γ-tubulin, to identify the importance of CUL3 in regulating mitosis. Furthermore, we have performed CUL3 immunoprecipitation (IP) experiments in X.laevis mitotic egg extracts and in HeLa cells synchronized at mitosis, to identify the binding partners of CUL3 which orchestrate mitosis. Using treatment with either nocodazole (promotes microtubule depolymerization) or monastrol (causes microtubule stabilization), we performed interaction studies in HeLa cells to determine the effect of microtubule polymerization on the interaction of CUL3 with its mitotic binding partners. Similarly, in X.laevis egg extracts, we have used warm (for microtubule depolymerization) and cold (for microtubule stabilization) IP conditions to determine the effects of microtubule status on CUL3 interaction with its binding partners. Our results have shown that CUL3-depleted cells display a dramatic mitotic phenotype which includes: defective spindles, multiple centrosomes, enlarged cells and nuclei, G2/M accumulation and delayed cytokinesis. To understand how CUL3 may participate in the formation of the mitotic spindle, we determined the binding partners of CUL3 that are involved in spindle assembly. CUL3 IP results from both X.laevis egg extracts and HeLa cells indicate that CUL3 interacts with the kinesin motor protein, Eg5. Eg5 is an important spindle assembly factor, which is essential in mitosis for proper formation of a bipolar spindle. Interestingly, targeting Eg5 is emerging as an attractive means to inhibit the proliferation of cancer cells. Thus, better characterizing the interaction of CUL3 and Eg5 is critical to understanding the function of CUL3 as well as Eg5 in mitosis. Further characterization of the interaction between CUL3 and Eg5 demonstrated that this interaction is present only in the presence of depolymerized microtubules. Our results culminate to the following conclusions: CUL3 is required for cells to properly undergo mitosis, especially the correct formation of spindle apparatus. CUL3 interacts with the spindle assembly factor Eg5 in the presence of depolymerized microtubules, specifically during mitosis. We suspect that CUL3-Eg5 interaction is important for mitosis and in particular for bipolar spindle formation. Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-288. doi:1538-7445.AM2012-LB-288