In the development of central nervous system (CNS)-targeted drugs, the prediction of human CNS target exposure is a big challenge. Cerebrospinal fluid (CSF) concentrations have often been suggested as a 'good enough' surrogate for brain extracellular fluid (brainECF, brain target site) concentrations in humans. However, brain anatomy and physiology indicates prudence. We have applied a multiple microdialysis probe approach in rats, for continuous measurement and direct comparison of quinidine kinetics in brainECF, CSF, and plasma. The data obtained indicated important differences between brainECF and CSF kinetics, with brainECF kinetics being most sensitive to P-gp inhibition. To describe the data we developed a systems-based pharmacokinetic model. Our findings indicated that: (1) brainECF- and CSF-to-unbound plasma AUC0-360 ratios were all over 100 %; (2) P-gp also restricts brain intracellular exposure; (3) a direct transport route of quinidine from plasma to brain cells exists; (4) P-gp-mediated efflux of quinidine at the blood-brain barrier seems to result of combined efflux enhancement and influx hindrance; (5) P-gp at the blood-CSF barrier either functions as an efflux transporter or is not functioning at all. It is concluded that in parallel obtained data on unbound brainECF, CSF and plasma concentrations, under dynamic conditions, is a complex but most valid approach to reveal the mechanisms underlying the relationship between brainECF and CSF concentrations. This relationship is significantly influenced by activity of P-gp. Therefore, information on functionality of P-gp is required for the prediction of human brain target site concentrations of P-gp substrates on the basis of human CSF concentrations.