Mitoxantrone (MXR) is a prototypical substrate for the ABCG2 (bcrp1) efflux transporter and is often used as a positive control in bcrp-mediated transport studies. This study examined processes involved in the cellular accumulation and directional transport of MXR using an in vitro wild-type (WT) and bcrp1-transfected (bcrp1) MDCKII cell model. Compared to 37 degrees C, incubation at 4 degrees C increased MXR accumulation in bcrp1 cells and unexpectedly decreased MXR accumulation in WT MDCKII cells. [3H]-MXR accumulation was concentration dependent in both WT and bcrp1 cells, exhibiting the characteristics of saturable active influx. At tracer concentrations, there was no difference in MXR directional flux between WT and bcrp1 MDCKII cell monolayers, and the A-to-B (apical-to-basolateral) flux was greater than the B-to-A flux in both cell types. However at higher concentration (20 microM), [3H]-MXR directional flux from A to B decreased and B to A increased, revealing the expected efflux process. Therefore, the orientation of the MXR directional flux process is concentration dependent and only at higher concentrations could the difference between WT and bcrp1 MDCKII cells be distinguished. Taken together, these data show that there is a saturable influx transport system on the apical membrane MDCKII cells that is responsible for the active influx of MXR. This is the first report of an active influx transport system for MXR. The expression of the putative MXR influx transporter in selected cell types could lead to misleading results in drug transport assays that screen for bcrp activity. Moreover, the downregulation of the influx transport system could be a heretofore unrecognized mechanism of MXR resistance in tumor cells.