Cytochrome bc 1 is a proven drug target in the prevention and treatment of malaria. The rise in drug-resistant strains of Plasmodium falciparum, the organism responsible for malaria, has generated a global effort in designing new classes of drugs. Much of the design/redesign work on overcoming this resistance has been focused on compounds that are presumed to bind the Q o site (one of two potential binding sites within cytochrome bc 1 ) using the known crystal structure of this large membrane-bound macromolecular complex via in silico modeling. Cocrystallization of the cytochrome bc 1 complex with the 4(1H)-pyridone class of inhibitors, GSK932121 and GW844520, that have been shown to be potent antimalarial agents in vivo, revealed that these inhibitors do not bind at the Q o site but bind at the Q i site. The discovery that these compounds bind at the Q i site may provide a molecular explanation for the cardiotoxicity and eventual failure of GSK932121 in phase-1 clinical trial and highlight the need for direct experimental observation of a compound bound to a target site before chemical optimization and development for clinical trials. The binding of the 4(1H)-pyridone class of inhibitors to Q i also explains the ability of this class to overcome parasite Q o -based atovaquone resistance and provides critical structural information for future design of new selective compounds with improved safety profiles. malaria | cytochrome bc 1 | drug discovery | Plasmodium falciparum | membrane protein