The function of breast cancer resistance protein (BCRP) and its role in drug absorption, distribution, and elimination has recently been evaluated. The objective of the present study was to examine the expression, localization, and functional characteristics of BCRP in Caco-2 cells, a widely used human intestinal epithelial cell model for investigating intestinal drug absorption. The expression of BCRP in Caco-2 cells was measured by Western blotting using the antibody BXP-21. Localization of BCRP was determined by an immunofluorescence technique using both antibodies BXP-21 and BXP-34. The drug efflux function of BCRP was evaluated via the epithelial transport of methotrexate (MTX) and estrone-3-sulfate (E3S) across Caco-2 cell monolayers in the presence or absence of the BCRP inhibitors Ko143 or GF120918 (N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide). Results from Western blot assay indicated that Caco-2 cells in the late passage (p56) expressed a higher level of BCRP as compared with the level in the early passages (p33). The total amount of BCRP protein did not change after the cells were confluent. Immunofluorescence studies revealed the positive staining of BCRP on the apical membrane of Caco-2 cells but not on the basolateral membrane after cell confluence. MTX and E3S showed a preferential basolateral-toapical (B-to-A) transport across Caco-2 cell monolayers. Both BCRP inhibitors Ko143 and GF120918 increased the apical-to-basolateral (A-to-B) transport but decreased the B-to-A transport of MTX and E3S. Caco-2 cells may therefore be used as an in vitro model to study the transport characteristics of BCRP.