Several membrane transporters belonging to the adenosine triphosphate-binding cassette (ABC) and solute carrier (SLC) families can transport drugs and drug metabolites and thereby exert an effect on drug absorption, distribution, and excretion, which may potentially lead to transporter-mediated drug-drug interactions (DDIs). Some transporter-mediated DDIs may lead to changes in organ distribution of drugs (eg, brain, liver, kidneys) without affecting plasma concentrations. Positron emission tomography (PET) is a noninvasive imaging method that allows studying of the distribution of radiolabeled drugs to different organs and tissues and is therefore the method of choice to quantitatively assess transporter-mediated DDIs on a tissue level. There are 2 approaches to how PET can be used in transporter-mediated DDI studies. When the drug of interest is a potential perpetrator of DDIs, it may be administered in unlabeled form to assess its influence on tissue distribution of a generic transporter-specific PET tracer (probe substrate). When the drug of interest is a potential victim of DDIs, it may be radiolabeled with carbon-11 or fluorine-18 and used in combination with a prototypical transporter inhibitor (eg, rifampicin). PET has already been used both in preclinical species and in humans to assess the effects of transporter-mediated DDIs on drug disposition in different organ systems, such as brain, liver, and kidneys, for which examples are given in the present review article. Given the growing importance of membrane transporters with respect to drug safety and efficacy, PET is expected to play an increasingly important role in future drug development.