Introduction <p>P-glycoprotein (P-gp) or multidrug-resistance protein is one of the most extensively studied efflux transporters at the blood-brain barrier (BBB). Changes in P-gp function are associated with several neurodegenerative and psychiatric diseases, including Alzheimer’s disease, Parkinson’s disease and schizophrenia and with the bioavailability of several pharmaceuticals in the brain, causing multi-drug resistance or side effects. PET imaging can be used to measure the P-gp function <i>in vivo.</i> This study aims to validate [¹⁸F]MC225 as specific P-gp PET tracer with the use of cyclosporin as selective P-gp inhibitor.</p> Methods <p>Fourteen healthy volunteers (age 67 ± 5y) were included. Subjects underwent twice a 60&#xa0;min dynamic [¹⁸F]MC225 (200MBq) PET scan with continuous arterial blood sampling and a cerebral T1-weighted MRI as anatomical reference. During the second scan, in five subjects, cyclosporin was administered in a dose of 2.5&#xa0;mg/kg/hour, starting 30&#xa0;min prior to the scan, to inhibit the BBB P-gp function. Tissue time-activity curves of preselected brain regions (Hammer’s atlas) were fitted to a reversible two-tissue compartment model (2T4k) using the metabolite corrected plasma and uncorrected whole blood curves as input functions.</p> Results <p>No significant difference was found in plasma kinetics, plasma curves, plasma-to-whole blood ratio, and the parent fraction of the baseline scans and scans after administration of cyclosporin. Volume of distribution values in whole brain grey matter showed a significant increase (6.18 ± 1.29 to 9.00 ± 1.29 mL·cm^− 3,<i>p</i> = 0.03) after the administration of cyclosporin.</p> Conclusion <p>The outcomes of the current study reflect the potential ability of [¹⁸F]MC225 to measure cyclosporin induced changes in P-gp function at the human BBB <i>in vivo.</i></p> Trial registration <p>EudraCT 2020-001564-28.</p>