Dichloroacetate (DCA) is a promising anticancer and antidiabetic compound targeting the mitochondrial pyruvate dehydrogenase kinase (PDHK). This study was undertaken in order to map the DCA-binding site of PDHK2. Here, we present evidence that R114, S83, I157 and, to some extent, H115 are essential for DCA binding. We also show that Y80 and D117 are required for the communication between the DCA-binding site and active site of PDHK2. These observations provide important insights into the mechanism of DCA action that may be useful for the design of new, more potent therapeutic compounds. © 2007 Federation of European Biochemical Societies.