The specificity and sensitivity of a flow cytometric assay simultaneously measuring expression and transport function of the multidrug resistance associated P-glycoprotein (Pgp) was evaluated. The monoclonal antibody (mAb), MRK16 was used to detect phenotypic Pgp expression while Fluo-3-AM was used as a fluorescent substrate in a Pgp functional transport assay. The specificity of the functional assay was examined in two vinblastine selected human leukemic cell lines (K562/VLB2.5 and CCRF-CEM/VLB50) with acquired Pgp overexpression. Downmodulation of Pgp function in these cell lines could be demonstrated with different substances (verapamil, vinblastine, trifluoperazine, cyclosporin A, progesterone and quinidine) and was proven to be consistently higher in the vinblastine selected cells than in their non-selected drug sensitive counterparts. Unexpectedly, modulator activity was also observed in drug sensitive K562 and CCRF-CEM cell lines despite the inability to detect Pgp in those cells by MRK16 flow cytometrically. Low level expression of the MDR1 gene encoding Pgp in sensitive K562 cells was however demonstrated with a sensitive RT-PCR procedure. The small effect of Pgp modulators in non-drug selected cells could therefore be attributed to low level basal expression of Pgp and illustrates the sensitivity of the functional assay. Also, the effect of various Pgp modulators on Pgp function was more pronounced in a subpopulation of Pgp expressing lymphocytes than in lymphocytes which did not express Pgp. Finally, a correlation was found between discrete variations in Pgp expression and Pgp function of CD4+ lymphocytes, underscoring the feasibility of the functional assay in a triple parametric procedure. The triple parametric assay holds promise to detect Pgp expression and function in clinical samples containing mixtures of malignant and non-malignant cells.