Multidrug resistance (MDR) in tumor cells is frequently associated with reduced cellular cytostatic drug accumulation, caused by the drug efflux protein, P-glycoprotein (Pgp). The action of Pgp in tumor cells can be detected by measuring the increase of daunorubicin accumulation upon blocking Pgp with drugs such as verapamil. A number of MDR cell lines have been described, characterized by decreased drug accumulation without Pgp being present. For such non-Pgp MDR cells no gene probes or functional assays are available to study this phenotype in clinical tumor specimens. We have worked out a method which enables the detection of drug-transport-related decreases in cellular daunorubicin accumulations without the need for the use of specific Pgp blockers. The cells used were SW-1573-, GLC4- and HT1080-sensitive cell lines, which accumulated (corrected for DNA content) 272%, 1,288% and 203% more daunorubicin than the non-Pgp MDR sublines SW-1573/2R120, GLC4/ADR and HT1080/DR4. When the plasma membranes of these MDR lines were permeabilized with 20 microM digitonin an increase to 282%, 1,260% and 239% of 14C-daunorubicin control accumulation was measured (at pH = 7.35). The intracellular pH measured with BCECF was the same in parent and corresponding MDR cells, excluding the role of pH differences in the measured effects. This method provides a tool allowing the detection of cellular mechanisms (including Pgp) which are related to active outward transport of daunorubicin.