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The chloroquine resistance transporter (PfCRT) confers resistance to a wide range of quinoline and quinoline-like antimalarial drugs in Plasmodium falciparum, with local drug histories driving its evolution and, hence, the drug transport specificities. For example, the change in prescription practice from chloroquine (CQ) to piperaquine (PPQ) in Southeast Asia has resulted in PfCRT variants that carry an additional mutation, leading to PPQ resistance and, concomitantly, to CQ re-sensitization. How this additional amino acid substitution guides such opposing changes in drug susceptibility is largely unclear. Here, we show by detailed kinetic analyses that both the CQ- and the PPQ-resistance conferring PfCRT variants can bind and transport both drugs. Surprisingly, the kinetic profiles revealed subtle yet significant differences, defining a threshold for in vivo CQ and PPQ resistance. Competition kinetics, together with docking and molecular dynamics simulations, show that the PfCRT variant from the Southeast Asian P. falciparum strain Dd2 can accept simultaneously both CQ and PPQ at distinct but allosterically interacting sites. Furthermore, combining existing mutations associated with PPQ resistance created a PfCRT isoform with unprecedented non-Michaelis-Menten kinetics and superior transport efficiency for both CQ and PPQ. Our study provides additional insights into the organization of the substrate binding cavity of PfCRT and, in addition, reveals perspectives for PfCRT variants with equal transport efficiencies for both PPQ and CQ. Author summary: Chloroquine (CQ) used to be the drug of choice against malaria until the parasite responsible for the disease became resistant. In the 1970s, piperaquine (PPQ) was introduced in areas where resistance to CQ was wide spread. In the following decade, an estimated 140 million doses were distributed, which substantially reduced the malaria burden, particularly in China, but created an environment in which PPQ resistant strains of the human malaria parasite Plasmodium falciparum emerged and spread. Interestingly, the PPQ resistant parasites displayed an increased CQ sensitivity. The main genetic determinant of both CQ and PPQ resistance in P. falciparum is a drug transporter, termed PfCRT. In this study, we used biochemical and bioinformatics approaches to understand how mutational changes in PfCRT influence the interaction of the carrier with CQ and PPQ. We found that PfCRT from CQ resistant parasites is better at transporting CQ than are PfCRT variants from PPQ resistant parasites, while the opposite is true for PPQ. We also found that PfCRT can be engineered such that it transports both antimalarials equally well. Our study offers insights into how PfCRT has evolved in response to changing drug pressure, and raises concerns regarding how it may evolve in the future. [ABSTRACT FROM AUTHOR]