Since March 1996, 93 consecutive samples of 45 adults and 41 children, suffering from acute leukemia, were evaluated prospectively for P-glycoprotein (P-gp) expression and function by flow cytometry. P-gp antigen expression was determined with the monoclonal antibodies 4E3 and MRK16. Transport function was assessed by measuring the modulating effect of verapamil on the intracellular retention of Rhodamine 123. P-gp positivity at relapse was not significantly more frequently than at initial diagnosis in our study group, neither with the immunologic assay, nor with the functional test. There was no correlation between the results of the immunologic and the functional test. Discordant test results were observed in 11/41 children (12/44 samples) and 14/45 adults (15/49 samples), independent of the type of leukemia. 2/12 and 2/15 samples scored positive with one of the monoclonal antibodies and were functionally inactive. In 10/12 and 13/15 samples however, an efflux pump was active and dependent of verapamil, but without detectable antigen. The functional flow cytometric assay allows evaluation of non-P-gp efflux pumps and, therefore, is more clinically relevant since it may better identify patients who would benefit from the use of P-gp inhibitors.