Chloroquine resistance in the malaria parasite Plasmodium falciparum is conferred by mutations in the P. falciparum chloroquine resistance transporter (PfCRT). PfCRT localizes to the membrane of the parasite's internal digestive vacuole, an acidic organelle in which chloroquine accumulates to high concentrations and exerts its toxic effect. Mutations in PfCRT are thought to reduce chloroquine accumulation in this organelle. How they do so is the subject of ongoing debate. Recently we have shown that in the presence of chloroquine there is an increased leak of H+ from the digestive vacuole in chloroquine-resistant but not chloroquine-sensitive parasites. Here, using transfectant parasite strains of a single genetic background and differing only in their pfcrt allele, we show that chloroquine resistance-conferring PfCRT mutations are responsible for this chloroquine-associated H+ leak. This is consistent with the hypothesis that the chloroquine resistance-conferring forms of PfCRT mediate the efflux of chloroquine, in association with H+, from the malaria parasite's digestive vacuole.