Cyclosporins are potent tools to inhibit several primary-active, ATP-dependent export carriers. This has been demonstrated in membrane vesicle transport assays for CsA and for its non-immunosuppressive analog PSC 833. Inhibition in the low micromolar and in the nanomolar concentration range is shown for the three distinct ATP-dependent export carriers in the liver canalicular membrane mediating the secretion into bile of leukotrienes (LTC4, other cysteinyl leukotrienes, and related conjugates), bile salts (taurocholate), and amphiphilic, mostly cationic substances (daunorubicin and other P-glycoprotein substrates). Competitive inhibition by cyclosporins is most potent for ATP-dependent taurocholate transport with Ki values of 0.2 and 0.6 microM for CsA and PSC 833, respectively. This inhibition is in agreement with in vivo studies in the rat demonstrating a block at the canalicular membrane in the hepatobiliary elimination of labeled taurocholate. The data suggest that cholestasis, as a side effect during CsA therapy, is largely due to inhibition of the ATP-dependent bile salt export carrier in the canalicular membrane. Inhibition by cyclosporins is less effective with respect to ATP-dependent leukotriene transport, both during biosynthetic release from mastocytoma cells and during hepatobiliary excretion. The Ki values for the former were 4.5 and 30 microM, and the Km/Ki ratios only 0.015 and 0.002 for CsA and PSC 833, respectively. Distinct transporters are inhibited by the cyclosporins with different potency and structurally modified cyclosporins may serve to induce preferential inhibition of a selected transporter. This is illustrated by the inhibition of the multidrug export carrier with daunorubicin as substrate using PSC 833 as inhibitor with a Ki value of 0.3 microM in an in vitro membrane transport system.