In an effort to characterize putative transport systems of valproic acid (VPA) at the blood-brain barrier, the effects of various substrates and inhibitors of known anion transporters on the equilibrium vessel-to-medium concentration (vessel/medium) ratio of VPA were investigated using isolated rat brain microvessels. The equilibrium vessel/medium ratio of VPA was decreased by the presence of high millimolar concentration of unlabeled VPA, indicating that a saturable transport system was involved in VPA transport from medium to microvessels. Short-chain monocarboxylates such as propionic acid, pyruvic acid, and L-lactic acid did not alter the vessel/medium ratio, whereas medium-chain fatty acids and unsaturated metabolites of VPA significantly inhibited the net transport of VPA. Dicarboxylates, tricarboxylate, and p-aminohippuric acid did not affect VPA accumulation in the brain microvessels. Several anionic drugs including salicylic acid, penicillin G, cefazolin, and probenecid significantly reduced the vessel/medium ratio of VPA. In addition, disulfonate inhibitors of inorganic anion exchangers, SH-group modifying reagent, and metabolic inhibitor showed remarkable inhibitory effects on the net transport of VPA between brain microvessels and medium. These results suggest that VPA may be actively transported through the antiluminal membrane via a carrier-mediated system shared by other anionic drugs.