To predict the magnitude of P-glycoprotein (P-gp)-based drug interactions at the human blood-brain barrier (BBB), rodent studies are routinely conducted where P-gp is chemically inhibited. For such studies to be predictive of interactions at the human BBB, the plasma concentration of the P-gp inhibitor must be comparable with that observed in the clinic. Therefore, we determined the in vivo EC(50) of P-gp inhibition at the rat BBB using verapamil as a model P-gp substrate and cyclosporine A (CsA) as the model P-gp inhibitor. Under isoflurane anesthesia, male Sprague-Dawley rats were administered i.v. CsA to achieve pseudo steady-state CsA blood concentrations ranging from 0 to approximately 12 microM. Then, an i.v. tracer dose of [(3)H]verapamil was administered, and 20 min after verapamil administration, the animals were sacrificed for determination of blood, plasma, and brain (3)H radioactivity by scintillation counting. The percentage increase in the brain/blood (3)H radioactivity (relative to 0 microM CsA) was described by the Hill equation with E(max), 1290%; EC(50), 7.2 microM; and gamma, 3.8. Previously, using [(11)C]verapamil, we have shown that the human brain/blood (11)C radioactivity was increased by 79% at 2.8 microM CsA blood concentration. At an equivalent CsA blood concentration, the rat brain/blood (3)H radioactivity was increased by a remarkably similar extent of 75%. This is the first time that an in vivo CsA EC(50) of P-gp inhibition at the rat BBB has been determined and the magnitude of such inhibition was compared between the rat and the human BBB at the same blood CsA concentration.