Human cytochrome P450 2D6 (CYP2D6) is responsible for the metabolism of approximately 20% of drugs in common clinical use. The CYP2D6 gene locus is highly polymorphic. Many of the polymorphisms have been shown to be clinically relevant and can account for inter-individual differences in the metabolism of specific drugs. In addition to the established sources of variability in CYP2D6-dependent drug metabolism, a recent study in our laboratory identified CYP2D6 in the mitochondria of human liver samples and found that it is metabolically active in this novel location. In the present study we show that mutations are present in the targeting signal region of CYP2D6 that may help to account for the inter-individual variability that was observed previously in the level of the mitochondrial enzyme in human liver samples. These mutations were identified within the ER targeting domain, the proline-rich domain as well as the putative protein kinase A (PKA) and protein kinase C (PKC)-specific phosphorylation sites. In vitro studies demonstrate that the mutations identified in the targeting signals affect the efficiency of mitochondrial targeting of CYP2D6. Since the mitochondrial enzyme has been shown to be active in drug metabolism, this pharmacogenetic variation could play a role in modulating the response of an individual to drug therapy.