BackgroundNormal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP‐binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux.MethodsWe used epifluorescence microscopy (EFM) in combination with single‐cell image analysis to study ABC‐transporter–mediated efflux in highly purified, viable, CD34+CD38‐ cells sorted on an adhesive biolayer. P‐glycoprotein and multidrug‐resistant protein (MRP)‐mediated efflux were quantitated using fluorescent substrates (rhodamine‐123 and calcein acetoxymethyl ester [calcein‐AM]) and specific inhibitors (verapamil and probenecid, respectively).ResultsThe feasibility, sensitivity, and reproducibility of rhodamine‐123 efflux quantitation using single‐cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P‐glycoprotein–mediated transport was higher in CD34+CD38‐ cells than in more differentiated progenitors (mean efflux index = 2.24 ± 0.35 and 1.14 ± 0.11, respectively; P = 0.01). P‐glycoprotein–mediated transport was the main determinant of the rhodamine “dull” phenotype of these cells. In addition, significant MRP‐mediated efflux was demonstrated in CD34+CD38‐ and CD38+ cells (mean efflux index = 1.42 ± 0.19 and 1.28 ± 0.18, respectively).ConclusionThe described method is a valuable tool for assessing ABC‐transporter–mediated efflux in highly purified single cells. Both P‐glycoprotein and MRP‐mediated efflux are present in human CD34+CD38‐ hematopoietic stem cells. Cytometry 49:135–142, 2002. © 2002 Wiley‐Liss, Inc.