Multidrug-resistant (MDR) cells are characterized by a defect in drug accumulation caused by overexpression of a transmembrane glycoprotein, the P-glycoprotein (P-gp). The MDR phenotype can be characterized either by use of monoclonal antibodies raised against P-gp or with functional tests based on the intracellular accumulation of fluorescent molecules. The aim of the present study was to compare the effectiveness of functional tests performed by flow cytometry including uptake of daunorubicin (DNR) (2 micrograms/ml), Hoechst 33342 (5 micrograms/ml), or rhodamine 123 (RH 123) (0.1 microgram/ml); and to evaluate the effect of cell death induced by heating at 60 degrees C for 2 h on incorporation of DNR and RH 123. Sensitive and resistant human hematopoietic K 562 cells expressing P-gp were identified by monoclonal antibodies C 219 and MRK-16. Fluorescence of the dyes was always higher in sensitive than in resistant cells. However, DNR and Hoechst 33342 produced a slight incorporation in resistant cells, while RH 123 showed lack of incorporation in resistant cells. Thus, RH 123 allows sensitive and resistant cells to be clearly distinguished. In case of cell death, accumulation of RH 123 and DNR were different. With RH 123, fluorescence intensity strongly decreased in sensitive cells. With DNR, fluorescence intensity was enhanced in resistant cells. Thus, when the MDR phenotype is defined by uptake of DNR or RH 123, artifactual results due to cell death may be avoided by using a dye such as propidium iodide to eliminate dead cells.