In cultured cells, curcumin (CUR) causes cell death by interfering with mitosis and leading to fragmented nuclei and disrupted microtubules, a process named mitotic catastrophe. In order to clarify the role of the known CUR metabolites hexahydro-CUR (HHC) and CUR-glucuronide (CUR-gluc) in mitotic catastrophe, the effects of CUR were studied in three human cancer cell lines with different metabolism of CUR. In Ishikawa and HepG2 cells, CUR was metabolized to HHC and small amounts of octahydro-CUR (OHC), whereas the only metabolism in HT29 cells was the formation of CUR-gluc. Despite their different metabolism, all three cell systems responded to CUR with arrest in G2/M phase and mitotic catastrophe. Fractionation of the cells showed that concentrations of CUR were higher in the ER and cytosol than in the incubation medium by a factor of up to about 150 and 8, respectively. In contrast to CUR, the metabolite HHC and the products of spontaneous degradation did not elicit any effects in Ishikawa cells. These results imply that the causative agent of mitotic catastrophe is the parent CUR molecule, whereas reductive metabolism and chemical degradation render CUR inactive.