One function of the vertebrate choroid plexus(CP) is removal of potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We have used confocal microscopy and quantitative image analysis to follow transport of the large organic anion, fluorescein-methotrexate (FL-MTX), from bath (CSF-side) to blood vessels in intact rat CP and found concentrative transport from CSF to blood. With 2 µM FL-MTX in the bath, steady state fluorescence in the subepithelium and vascular spaces exceeded bath levels by 5-10-fold, but fluorescence in epithelial cells was below bath levels. FLMTX accumulation in subepithelium and vascular spaces was reduced by NaCN, Na removal and by other organic anions, e.g., MTX, probenecid, and estrone sulfate. Increasing medium K 10- fold had no effect. None of these treatments affected cellular accumulation. However, two observations indicated that apical FL-MTX uptake was indeed mediated: first, cellular accumulation was a saturable function of medium substrate concentration; second, digoxin and MK571 reduced FL-MTX accumulation in the subepithelial/vascular spaces but also increased cellular accumulation several-fold. In the presence of digoxin and MK571, cellular accumulation was concentrative, specific and Na-dependent. Thus, transepithelial FL-MTX transport involved two mediated steps: Na-dependent uptake at the apical membrane and electroneutra efflux at the basolateral membrane, possibly on Oatp2 and Mrp1.