We have studied antifolate-resistant rodent cell lines with respect to dihydrofolate reductase gene expression and expression of the "classic" multidrug resistance phenotype by flow cytometry. Using a series of antifolate-resistant and colchicine-resistant Chinese hamster ovary cell lines obtained by single-step and stepwise selection protocols, we show that viable cell staining with fluoresceinated methotrexate and daunorubicin correlates well with drug resistance and expression of dihydrofolate reductase protein and the "classic" MDR phenotype in these cell lines. We show that flow cytometric analysis makes it possible to rapidly assess the potentially complex drug resistance phenotype(s) of cells selected with hydrophilic and lipophilic antifolates.