Although sequence changes in the malarial multidrug-resistance gene, Pfmdr1, such as asn86tyr, have been linked with chloroquine-resistance in a collection of routinely cultured, laboratory strains of Plasmodium falciparum, an experimental cross indicates that another gene may be involved. Mammalian cells transfected with wild-type Pfmdr1 are more susceptible to chloroquine but 3′ mutations abolish the effect by increasing the lysosomal pH, probably due to defective anion-channel activity. In malarial parasites, overexpression of wild-type Pfmdr1 leads to increased chloroquine sensitivity. Use of PCR amplification on uncultivated material from Zaria confirms that a highly significant, though incomplete, association exists between asn86tyr and resistance to chloroquine, supporting the involvement of this gene. If a Pfmdr1gene with a resistance-related mutation were overexpressed, this might have a different effect from overexpression of a wild-type gene. A functional or non-functional anion channel associated with the carboxy-terminal end of the Pfmdr1 product could lead to conflicting effects of overexpression, depending on the presence or absence of mutations associated with either end. It is possible that the additional gene involved in resistance may regulate baseline lysosomal pH, which could have a primary influence on resistance, irrespective of mutations in Pfmdr1.