OBJECTIVE Cytotoxic anticancer drugs are less effective in killing tumor cells grown as multicellular spheroids than monolayer cell cultures. The aim of this study was to investigate the molecular mechanism of chemoresistance. METHODS Ovarian cancer A2780, CAOV3 multicellular spheroids were obtained from three-dimensional culture. Expression of P-glycoprotein (P-gp) was determined using Western blot analysis and flow cytometry (FCM). The subcellular distribution of P-gp was also determined using laser confocal microscope. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect the mdr1 mRNA of both monolayer cells and multicellular spheroids. Cell cycle profiles and apoptosis were also analyzed using FACS. The resistance was detected with trypan blue exclusion testing. RESULTS Compared with control cells, no expression of P-gp was detected in monolayer cells, but expression of P-gp in aggregate cells was significantly elevated(P< 0.05). The mdr1 mRNA positive nodes were confirmed by RT-PCR in the aggregate cells. The percentage of cells increased in G(0)-G(1) phase and decreased in S and G(2)-M phase significantly in spheroids cells. Spheroids cells showed higher cell viability than monolayer cells (P(A2780)=0.003, P(CAOV3)=0.015). More apoptotic cells were induced by Taxol in MCS cells than in monolayer cells. CONCLUSIONS Ovarian cancer A2780, CAOV3 multicellular spheroids cultures induced cell resistance to Taxol. High expression of P-gp was induced in ovarian multicellular spheroids and the cells were arrested in G(0)-G(1) phase.