Most multidrug resistant cell lines reported in the literature were established by long-term continuous exposure of cells to stepwise increasing concentrations of antitumor drugs. However, these resistant cell lines may not be relevant to the majority of clinically resistant cells. In this study, we described the establishment of doxorubicin (Dox)-resistant Chinese hamster ovary cells by repeated flow cytometric cell sorting using the intrinsic fluorescence of Dox. In each sorting, the 15% least fluorescent cells were fractionated, grown to mass culture and sorted again. Results from a total of nine sorting cycles showed that the intracellular levels of Dox in the sorted cells were inversely proportional to the number of sorting cycles. The levels of P-glycoprotein mRNA in the sorted cells were increased, but reached a plateau of 2-3 fold after the fifth sorting cycle. The sorted cells exhibited a moderate but stable multidrug-resistant phenotype. Because the procedure involved minimal exposure of cells to the drug, the isolated cells are most likely related to naturally occurring (intrinsic) MDR cells.