Atovaquone is active in vitro against the tachyzoites of Toxoplasma gondii at nanomolar concentrations and is used clinically to treat acute cases of human toxoplasmosis. In pursuit of the mechanism of action of atovaquone against T. gondii and to understand how resistance might arise, drug-resistant mutants were generated and examined. The previously uncloned cytochrome b gene of T. gondii was cloned and sequenced from wild type and resistant strains as this was a likely candidate for the target of the drug and thus a source of resistance. Mutations are present within the cytochrome b gene of atovaquone-resistant parasites (M129L and I254L) and represent alterations in two different regions of the ubiquinol-binding pocket (Q(o) domain) of cytochrome b, suggesting that atovaquone interferes with electron transport at the cytochrome bc(1) complex in T. gondii. A structural model for how this hydroxynaphthoquinone is binding within the Q(o) domain is presented. Further analysis of the cytochrome b gene suggested that the protein may differ from other homologues by terminating within the mitochondrial membrane. Cytochrome b becomes the first complete mitochondrial gene and cognate protein to be described for T. gondii.