The ATP-driven xenobiotic transporter P-glycoprotein is a critical element of the blood-brain barrier. To study regulation of P-glycoprotein function, we measured specific transport [(3'-oxo-4-butenyl-4-methyl-threonine(1), (valine(2)) cyclosporin (PSC833)-sensitive] of the fluorescent cyclosporin A derivative [N-epsilon(4-nitrobenzofurazan-7-yl)-D-Lys(8)]-cyclosporin A (NBDL-CSA) into the lumens of isolated rat brain capillaries using confocal microscopy and quantitative image analysis. Luminal NBDL-CSA accumulation was rapidly and reversibly reduced in a concentration-dependent manner by 0.1 to 100 nM endothelin-1 (ET-1). In this concentration range, ET-1 did not affect junctional permeability. The ET(B) receptor agonist sarafotoxin 6c also reduced transport. An ET(B) receptor antagonist blocked effects of ET-1 and sarafotoxin 6c; an ET(A) receptor antagonist was without effect. Consistent with this, immunostaining and Western blotting showed expression of the ET(B) receptor in brain capillary membranes. NBDL-CSA transport was also reduced by sodium nitroprusside, a NO donor, and by phorbol ester, a protein kinase C (PKC) activator. Inhibition of NO synthase (NOS) or PKC abolished the ET-1 effects. Thus, ET-1, acting through an ET(B) receptor, NOS, and PKC rapidly and reversibly reduced transport mediated by P-glycoprotein at the blood-brain barrier.