Ko143 is a reference inhibitor of the adenosine triphosphate-binding cassette (ABC) transporter breast cancer resistance protein (humans: ABCG2, rodents: Abcg2) for in vitro and in vivo use. Previous in vitro data indicate that Ko143 binds specifically to ABCG2/Abcg2, suggesting a potential utility of Ko143 as a positron emission tomography (PET) tracer to assess the density (abundance) of ABCG2 in different tissues. In this work we radiolabeled Ko143 with carbon-11 (11C) and performed small-animal PET experiments with [11C]Ko143 in wild-type, Abcg2(-/-), Abcb1a/b(-/-) and Abcb1a/b(-/-)Abcg2(-/-) mice to assess the influence of Abcg2 and Abcb1a/b on tissue distribution and excretion of [11C]Ko143. [11C]Ko143 was extensively metabolized in vivo and unidentified radiolabeled metabolites were found in all investigated tissues. We detected no significant differences between wild-type and Abcg2(-/-) mice in the distribution of [11C]Ko143-derived radioactivity to Abcg2-expressing organs (brain, liver and kidney). [11C]Ko143 and possibly its radiolabeled metabolites were transported by Abcb1a and not by Abcg2 at the mouse blood-brain barrier. [11C]Ko143-derived radioactivity underwent both hepatobiliary and urinary excretion, with Abcg2 playing a possible role in mediating the transport of radiolabeled metabolites of [11C]Ko143 from the kidney into urine. Experiments in which a pharmacologic dose of unlabeled Ko143 (10 mg/kg) was co-administered with [11C]Ko143 revealed pronounced effects of the vehicle used for Ko143 formulation (containing polyethylene glycol 300 and polysorbate 80) on radioactivity distribution to the brain and the liver, as well as on hepatobiliary and urinary excretion of radioactivity. Our results highlight the challenges associated with the development of PET tracers for ABC transporters and emphasize that inhibitory effects of pharmaceutical excipients on membrane transporters need to be considered when performing in vivo drug-drug interaction studies. Finally, our study illustrates the power of small-animal PET to assess the interaction of drug molecules with membrane transporters on a whole body level.