Breast cancer resistance protein (BCRP), an ATP-dependent efflux transporter, confers drug resistance to many chemotherapy agents. BCRP is overexpressed in tumors exposed to an acidic environment; therefore, it is important to establish the effect of low pH on BCRP transport activity. It has recently been reported that BCRP transports substrates more efficiently in an acidic microenvironment. In the study presented here, we examine the pH dependence of BCRP using methothrexate (MTX), pemetrexed (PMX), and estrone sulfate (ES) as model substrates. Our study revealed an increase of approximately 40-fold in the BCRP-mediated transport of PMX and MTX when the pH was decreased from 7.4 to 5.5. In contrast, only a 2-fold increase was observed for ES. These results indicate a mechanism of transport that is directly dependent on the effective ionization state of the substrates and BCRP. For ES, which retains a constant ionization state throughout the applied pH, the observed mild increase in activity is attributable to the overall changes in the effective ionization state and conformation of BCRP. For MTX and PMX, the marked increase in BCRP transport activity was likely due to the change in ionization state of MTX and PMX at lowered pH and their intermolecular interactions with BCRP. To further rationalize the molecular basis of the pH dependence, molecular modeling and docking studies were carried out using a homology model of BCRP, which has previously been closely examined in structural and site-directed mutagenesis studies (Am J Physiol Cell Physiol 299:C1100-C1109, 2010). On the basis of docking studies, all model compounds were found to associate with arginine 482 (Arg482) by direct salt-bridge interactions via their negatively charged carboxylate or sulfate groups. However, at lower pH, protonated MTX and PMX formed an additional salt-bridge interaction between their positively charged moieties and the nearby negatively charged aspartic acid 477 (Asp477) carboxylate side chain. The formation of this "salt-bridge triad" is expected to increase the overall electrostatic interactions between MTX and PMX with BCRP, which can form a rational basis for the pH dependence of the observed enhanced binding selectivity and transport activity. Removal of Arg482 in site-directed mutagenesis studies eliminated this pH dependence, which lends further support to our binding model. These results shed light on the importance of electrostatic interactions in transport activity and may have important implications in the design of ionizable chemotherapeutics intended for tumors in the acidic microenvironment.