Multidrug resistance (MDR) is often related to expression of P-glycoprotein (Pgp) or Multihg Resistance Protein (MRP). Pgp-mediated MDR can be evaluated by determining cellular retention of fluorescent substrates by flow cytometry. This study determined if agents used to evaluate Pgp function also can be used to evaluate MRP function. Cellular retention of doxorubicin (Dox), Rhodamhe-123 (Rh-123), and 3 , 3 ' -d i e t h y l o x a c a r ~e iodide (DiOC2(3)) were studied i n MRP-expressing cell lines (HLGO/Adr and HTlOSO/DR4), whereas a Pgp expressing cell line (A2780/Dx5) served as a positive control. Overexpression of Pgp correlated inversely with retention of Dox, Rh-123, and DiOC,(3); however, under identical experimental conditions (1 h reincubation in drug-free medium), no retention difference of the three agents was detected between parental and MRP-expressing resistant cells. Upon extending the reincubation time to 4 h, an efflux of Rh-123 and Dox in the resistant h e s became apparent and even more pronounced after 24 h; however, still no efflux was detectable for DiOC2(3). Incubation of the cells with a modulator of MDR, PAK-104P, negated the observed drug efflux in Pgp and MRP expressing cells, which correlated with increased sensitivity of the MDR lines to doxorubicin. Thus both Dox and Rh-123 can be used to evaluate MRP-function, but DiOC,(3) can not.