The p-glycoprotein is a transmembrane efflux transporter found on the luminal side of the capillary endothelial cells that comprise the blood-brain barrier. This study examined the effect of a p-glycoprotein inhibitor, cyclosporin A, on the distribution to the brain of a p-glycoprotein substrate, rhodamine-123, in freely moving rats using intracerebral microdialysis coupled with on-line HPLC analysis. Results from crossover experiments show that the coadministration of cyclosporin A significantly increased the distribution of rhodamine-123 to the brain. The plasma disposition of rhodamine-123 was unchanged by cyclosporin A, indicating that the change in brain exposure was mediated by a process at the level of the blood-brain barrier, possibly by inhibition of the p-glycoprotein efflux transporter. This finding suggests a functional activity of the p-glycoprotein in the blood-brain barrier and validates an in vivo model to examine the role of this transporter in the brain distribution of drugs.