Fluorescent dyes such as rhodamine 123 (R123) and Hoechst 33342 (Ho342) have been widely used to characterize multidrug-resistance (MDR) phenotype cells in cell populations, on the basis of their reduced accumulation in resistant cells. Taking advantage of the high fluorescence quantum yield of R123 and Ho342 compared with that of anthracyclines, we investigated the limits of fluorescence image cytometry in detecting MDR by the level of R123 and Ho342 accumulation and efflux. We were able to separate with this technique a cell line with a level of resistance as low as 3. We then studied the presence of MDR cells in lymphocytes isolated from patients with hematological malignancies.