The expression of CD133 is strictly limited to a rare population of somatic and cancer stem cells. It is therefore difficult to obtain sufficient numbers of cells to perform biochemical analysis of the CD133-containing protein complex. Taking advantage of the capability of CCC stem cells to grow exponentially and maintain high expression levels of CD133 in vitro, we set out to immunopurify the endogenous CD133 complex. CD133 was immunoprecipitated from the membrane fraction with antiCD133 antibody and after confirmation by SDS-PAGE and silver staining, the immunoprecipitates were subjected to liquid chromatography-mass spectrometry. Among the co-purified proteins identified, we focused our attention on plakoglobin and desmoplakin, since they are components of the desmosome, which mediates cell-cell adhesion. Desmosomes are junctional complexes consisting of members of the cadherin family of cell adhesion proteins and linking proteins that attach the cell surface adhesion proteins to intracellular keratin cytoskeletal filaments. Plakoglobin and desmoplakin function as the main desmosomal linking proteins. We confirmed the ability of CD133 to interact with plakoglobin by in vivo pull-down assays. When a lysate from CCC stem cells was subjected to immunoprecipitation with anti-CD133 antibody, followed by immunoblotting with anti-plakoglobin antibody, plakoglobin was found to have co-immunoprecipitated with CD133. Plakoglobin was not detected when control IgG was used for immunoprecipitation. However, our in vitro pulldown assays failed to detect co-precipitation of plakoglobin with fragments containing individual cytoplasmic domains of CD133. This may be because the membrane topology of CD133 is important for its association with plakoglobin. Alternatively, CD133 may not be directly associated with plakoglobin. Results with desmoplakin were inconclusive, as it co-precipitated with either the anti-CD133 antibody or control IgG under our experimental conditions. Immunohistochemical analysis of CCC stem cells revealed that CD133 and plakoglobin co-localized within regions of cell-cell contact. CD133 staining was not detected when cells were infected with a lentivirus expressing an shRNA targeting CD133, indicating the specificity of anti-CD133 antibody. Desmoplakin was found to partially co-localize with CD133. We performed immunohistochemical analysis of desmoglein-2 and desmocollin-2, two desmosomal cadherins that are expressed in CCC stem cells. We found that these proteins also co-localized with CD133. In particular, CD133 and desmoglein-2 had very similar distribution patterns. However, neither desmoglein-2 nor desmocollin-2 could be detected in CD133 immunoprecipitates, indicating that they are not physically associated. By contrast, plakoglobin immunoprecipitates were found to contain CD133 as well as desmosomal cadherins. Altogether, these results suggest that CD133 MicroRNAs likely influence these processes by negative regulation through binding to messenger RNA targets interacts with plakoglobin but not with the desmosomal protein complex containing desmoglein-2 and desmocollin-2. We next studied the role of CD133 in the regulation of cell-cell adhesion. We observed that CCC stem cells could not be readily dispersed by pipetting. However, when cells were infected with a lentivirus expressing an shRNA targeting CD133, the cells could be dispersed by pipetting. Moreover, hanging drop cell aggregation assays demonstrated that CD133 knockdown cells did not aggregate tightly and could be dispersed by pipetting. Thus, CD133 may be important for the adhesion of CCC stem cells.