We had previously shown that doxorubicin encapsulation in polyisohexylcyanocrylate nanospheres could circumvent the P-glycoprotein-mediated multidrug resistance (MDR) exhibited by C6 rat glioblastoma in culture. We then investigated what could be the mechanism of such a circumvention. The cytotoxicity of free and encapsulated doxorubicin was evaluated in two MDR variants of the C6 cell line in a device allowing the separation of cells from drugs by a polycarbonate membrane of 0.2 micron pore size. We observed that the progressive disruption of the nanospheres allowed their doxorubicin content to reach the cell monolayer and exert its cytotoxicity in a fashion similar to that exhibited by free doxorubicin. However, no circumvention of MDR is obtained by doxorubicin encapsulation when drug-containing nanospheres are separated from the cells by the polycarbonate membrane. In addition, no effect on azidopine binding to P-glycoprotein-enriched membranes is exerted by unloaded nanospheres, even after their spontaneous degradation in cell-culture medium. Taken together, these results suggest that a physical contact between doxorubicin-containing nanospheres and the cells is required for the circumvention of MDR. The role of degradation products from the nanospheres as modulators of P-glycoprotein activity can be ruled out.