Three-dimensional cell cultures (spheroids) provide an in vitro approximation of solid tumors in vivo. Some mechanisms of drug resistance were reported to differ in their effects on monolayer and spheroid cells. We have compared the effect on the expression of P-glycoprotein (Pgp), the MDR1 gene product, on vinblastine resistance in two glioblastoma lines, U87 and U251, grown as either monolayers or spheroids. U87 cells form spheroids spontaneously and the spheroids continue to grow, while U251 cells form spheroids only when plated over agarose and do not proliferate in spheroids. U87 cells are equally resistant to vinblastine in monolayer and in spheroids, while U251 cells are 4.5 times more resistant in spheroids than in monolayers. The distribution of a fluorescent vinblastine derivative in cells of spheroids was more heterogeneous than in monolayers. MDR1-expressing U87 cells increased their resistance 9-fold in monolayer and 50-fold in spheroids, while MDR1 transduction of U251 cells made them 20-fold more resistant in monolayer and 160-fold more resistant in spheroids. MDR1 expression in both cell lines decreased the accumulation of 3H-vinblastine and fluorescent vinblastine derivative. These results indicate that MDR1 is more efficient in conferring drug resistance in spheroids than in monolayer cells.