Multidrug resistant cancer cells of the MDR-1 phenotype utilize an ATP-dependent pump to excrete toxic drugs. Rhodamine 123 (R123) is a fluorescent substrate of the MDR pump. An assay for the ATP-dependent initial efflux of R123 from CEM/VLB100 human leukemic lymphoblasts has been developed. The MDR-1 cells were treated with a reversal agent and preloaded with 40.0 nM R123 in buffer at 30 degrees C that contained sodium azide and 2-deoxyglucose. The cells were rinsed with cold buffer and resuspended in L-glutamine/glucose solution at 23 degrees C. The cell suspension was passed through a filter and R123 in the filtrate was detected at 2-s intervals by fluorescence. Efflux of R123 was inhibited by the reversal agents amiodarone, cyclosporin A, Ro11-2933 (DMDP), quinidine, and the optical isomers of propranolol. The MDR pump is stereospecific for the (R)-diastereomer quinidine; however, the (S)-diastereomer quinine is a relatively weak inhibitor of the pump. Cyclosporin A was the most potent inhibitor tested against the efflux of R123 by the MDR pump.