The genetic basis of variation in resistance to natural toxins is of interest for both ecological and evolutionary genetics. The wide variety of larval resources used by Drosophila, both within and between species, makes flies an excellent system for studying causes and consequences of selection resulting from exposure to natural toxins associated with different resources. In this study we carry out a genetic analysis of α-amanitin resistance in a population sample of Drosophila melanogaster. Data from mapping crosses of chromosome III support a role for a naturally occurring polymorphism in a multidrug resistance gene (Mdr65A) in α-amanitin resistance. However, there are no amino acid differences between resistant and sensitive chromosomes at Mdr65A. Therefore, if Mdr65A mutants contribute to the difference between α-amanitin-resistant and α-amanitin-sensitive third chromosome lines, the underlying cause is a gene regulatory mutation.