This article describes the experimental set-up and pharmacokinetic modeling of P-glycoprotein function in the rat blood-brain barrier using [ 11 C]verapamil as the substrate and cyclosporin A as an inhibitor of P-gp. [ 11 C]verapamil was administered to rats as an i.v. bolus dose followed by graded infusions to obtain steady-state concentrations in the brain during 70 min. CsA was administered as a bolus followed by a constant infusion 20 min after the start of the [ 11 C]verapamil infusion. The brain uptake of [ 11 C]verapamil over 2 h was portrayed in a sequence of PET scans in parallel with measurement of [ 11 C]verapamil concentrations in blood and plasma and CsA concentrations in blood. Mixed effects modeling in NONMEM was used to build a pharmacokinetic model of CsA-induced P-gp inhibition. The brain pharmacokinetics of [ 11 C]verapamil was well described by a two-compartment model. The effect of CsA on the uptake of [ 11 C]verapamil in the brain was best described by an inhibitory indirect effect model with an effect on the transport of [ 11 C]verapamil out of the brain. The CsA concentration required to obtain 50% of the maximal inhibition was 4.9 mg/mL (4.1 mM). The model parameters indicated that 93% of the outward transport of [ 11 C]verapamil was P-gp mediated.