Flotillins are membrane association proteins consisting of two homologous members, flotillin-1 (Flot-1) and flotillin-2 (Flot-2). They define a clathrin-independent endocytic pathway in mammal cells, which are also distinct from some other endocytosis mechanisms. The implicated cargoes of the flotillin-dependent pathway are mainly some GPI-anchored proteins, such as CD59 and Thy-1, which positionally colocalize with flotillins at the plasma membrane microdomains. To see whether flotillins are involved in the endocytosis of PrP(C), the potential molecular interaction between PrP(C) and flotillins in a neuroblastoma cell line SK-N-SH was analyzed. Co-immunoprecipitation assays did not reveal a detectable complex in the cell lysates of a normal feeding situation. After stimulation of Cu(2+), PrP(C) formed a clear complex with Flot-1, but not with Flot-2. Immunofluorescent assays illustrated that PrP(C) colocalized well with Flot-1, and the complexes of PrP(C)-Flot-1 shifted from the cell membrane to the cytoplasm along with the treatment of Cu(2+). Down-regulating the expression of Flot-1 in SK-N-SH cells by Flot-1-specific RNAi obviously abolished the Cu(2+)-stimulated endocytosis process of PrP(C). Moreover, we also found that in the cell line human embryonic kidney 293 (HEK293) without detectable PrP(C) expression, the distribution of cellular Flot-1 maintained almost unchanged during Cu(2+) treatment. Cu(2+)-induced PrP(C)-Flot-1 molecular interaction and endocytosis in HEK293 cells were obtained when expressing wild-type human PrP (PrP(PG5)), but not in the preparation expressing octarepeat-deleted PrP (PrP(PG0)). Our data here provide direct evidences for the molecular interaction and endocytosis of PrP(C) with Flot-1 in the presence of copper ions, and the octarepeat region of PrP(C) is critical for this process, which strongly indicates that the Flot-1-dependent endocytic pathway seems to mediate the endocytosis process of PrP(C) in the special situation.