Multidrug and toxin extrusion protein 1 (MATE1) and MATE2-K are organic cation/H(+) antiporters that have recently been identified and suggested to be responsible for the brush border secretory transport of many cationic drugs in renal tubules. We here report our finding that 4',6-diamidino-2-phenylindole (DAPI) can be used as a probe substrate for rapid assays of the functionality of the human MATEs, hMATE1, and hMATE2-K, by taking advantage of its fluorescent nature. The specific cellular uptakes of DAPI by cloned hMATE1 and hMATE2-K, which were assessed by fluorescence intensity, were found to be rapid and saturable with the Michaelis constants of 1.13 and 3.16 microM, respectively, indicating that DAPI is a good substrate of both hMATEs. It was found that many organic cations inhibit the specific uptake of DAPI by hMATE1 and hMATE2-K, and the extents of inhibition are in good correlation with those of inhibition of the specific uptake of [(3)H]cimetidine as a typical substrate, indicating comparable performances of both substrates as probes in identifying inhibitors. Thus, DAPI can be an alternative probe substrate that enables fluorometric rapid assays of the functionality of both hMATEs. It was also found that the other major renal organic cation transporters, human organic cation transporter 2 (hOCT2), hOCT3, human novel organic cation transporter 1 (hOCTN1), and hOCTN2, cannot transport DAPI, although hOCT1, which is mainly expressed in the liver, can. Therefore, the DAPI uptake assay can be a method specific to the hMATEs among organic cation transporters in the human kidney.