Plasmodium falciparum isolates with decreased susceptibility to quinine are increasingly being found in malaria patients. Mechanisms involved in this resistance are not yet understood. Several studies claim that alongside mutations in the Pfcrt and Pfmdr1 genes, the Pfnhe-1 Na(+)/H(+) exchanger polymorphism plays a role in decreasing susceptibility. However, conflicting results on the link between the Pfnhe-1 gene and quinine resistance arise from field- and culture-adapted isolates. We tested the association between Pfnhe-1, Pfcrt, and Pfmdr1 polymorphisms in field- and culture-adapted isolates from various countries with their in vitro susceptibility to quinine. Field isolates presented a higher diversity of the Pfnhe-1 microsatellite sequence than culture-adapted isolates. In culture-adapted isolates but not in field isolates, mutations in the Pfcrt and Pfmdr1 genes, as well as a higher number of DNNND repeats in the Pfnhe-1 gene, were associated with a higher 50% inhibitory concentration (IC(50)) of quinine. Furthermore, most of the culture-adapted isolates with more than one DNNND repeat in the Pfnhe-1 gene also harbored mutated Pfcrt and Pfmdr1 genes with an apparent cumulative effect on quinine susceptibility. This study supports the involvement of the Pfnhe-1 gene in the modulation of the in vitro quinine response when associated with mutated Pfcrt and Pfmdr1 genes. Culture adaptation could be responsible for selection of specific haplotypes of these three genes. Methods used for drug testing might thus influence the association between Pfnhe-1 polymorphism and quinine susceptibility. However, we do not exclude the possibility that in particular settings, Pfnhe-1 polymorphism can be used as a molecular marker for surveillance of quinine resistance.