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Background: Multidrug resistance is the most frequent type of resistance to anticancer chemotherapy and diseasemodifying antirheumatic drugs (DMARDs), which usually results from the overexpression of efflux transporters,such as the MDR1, MRP1 and BCRP.MultiDrugQuant (MDQ) kit was developed as an improved functional assay system, which can measure the multidrug resistance activity of the three, clinically most relevant efflux transporters using flow cytometry in living cells.The present study aimed to carry out the laboratory validation and to evaluate the performance of the MDQ-kit according to the requirements to be applied for routine diagnostic methods.Methods: Validation of the kit was carried out according to the standards of the Clinical Laboratory Standards Institute in three university centres.Mononuclear cells from 128 normal volunteers and from 60 patients (CLL and AML) were separated by Ficoll gradient and labeled by CD19 to study B-CLL cells and CD45 do identify CD45-dim AML blasts.Flow cytometric measurements were carried out at 2-5x 106 cells/ml within 6 hours after specimen collection.Activities of the multidrug transporters were calculated from the difference between the mean fluorescent intensity of cells w/o the specific inhibitors, respectively.Inaccuracy and comparative measurements were carried out using MDR1 or MRP1 positive and negative HL60 cells and BCRP positive or negative PLB cell lines with low and high activity of the transporters.Results: The specificity and robustness of the assay have been demonstrated at different concentrations of the fluorescent dyes (10-100 % of the original) or inhibitors (50-150 % of the original).Intraassay and interassay reproducibilities of the tests were <5%.Multidrug resistance activity values determined on different flow cytometers were comparable and eligible.Conclusions: The MDQ assay provides quantitative results on the activity of the MDR 1, MRP 1 and BCRP in the target cells, which might be used to predict the resistance of these cells to particular cytotoxic agents.Recently, the MDQ-kit has been registered for in vitro diagnostic use in the EU.