The development of multidrug resistance (MDR) in heterogeneous cell sensitive and resistant populations to a variety of clinically important cytotoxic drugs poses a major obstacle to cancer chemotherapy. The MDR phenotype is characterized by a decrease the intracellular drug accumulation and by an overexpression of the MDR1 gene which encodes the membrane protein, P-glycoprotein (Pgp). To evaluate the MDR phenotype, rationale investigations of the cytotoxic processes and effect,s of Adriamycin (ADR) were done to obtain information on individual cells. Such information could be obtained through a multiparametric approach involving multiwavelength microfluorometry and numerical image analysis on single living cells. To achieve this, cells should be simultaneously stained with Hoechst 33342 (nuclear staining), Rhodamine 123 (mitochondria staining) and Nile Red (cell contour delineation). Changes in the biological parameters accessible from R123, Ho33342 and C-SNARF-1/AM (probe used for the pHi measurements) labelling were found more informative than changes in morphological parameters for the discrimination of sensitive and resistant cells. Furthermore, this approach allows the discrimination between two resistant cell lines expressing different mechanisms of resistance.