Delivery of valproic acid (VPA) to the human brain is relatively inefficient as reflected by a low brain-to-unbound plasma concentration ratio (< or =0.5) at steady state. Previous pharmacokinetic studies suggested that the unfavorable brain-to-plasma gradient is maintained by coupled efflux transport processes at both the brain parenchymal cells and blood-brain barrier (BBB); one or both of the efflux transporters are inhibitable by probenecid. The present study in rabbits utilized microdialysis to measure drug concentration in the brain extracellular fluid (ECF) of the cerebral cortex during steady-state i.v. infusion with VPA alone or with VPA plus probenecid. Probenecid co-infusion elevated VPA concentration in the brain tissue surrounding the tip of the microdialysis probe to a greater extent than in the ECF (230% versus 47%). Brain intracellular compartment (ICC) concentration was estimated. In control rabbits, the ICC concentration was 2.8+/-0.28 times higher than the ECF concentration. Probenecid co-infusion elevated the ICC-to-ECF concentration ratio to 4.2+/-0.44, which confirms the existence of an efflux transport system in brain parenchymal cells. The ECF-to-unbound plasma concentration ratio was well below unity (0.029), indicating an uphill efflux transport of VPA across the BBB. Co-infusion of probenecid did not have a significant effect on VPA efflux at the BBB as evidenced by a minimal change in the ECF-to-unbound plasma concentration ratio. This study suggests the presence of distinctly different organic anion transporters for the efflux of VPA at the parenchymal cells and capillary endothelium in the brain.