1. P-glycoprotein, a 170-180 kDa membrane glycoprotein that mediates multidrug resistance, hydrolyses ATP to efflux a broad spectrum of hydrophobic agents. In this study, we analysed the effects of three MDR reversing agents, verapamil, cyclosporin A and [3'-keto-Bmt1]-[Val2]-cyclosporin (PSC 833), on the adenosine triphosphatase (ATPase) activity of human P-glycoprotein. 2. P-glycoprotein was immunoprecipitated with a monoclonal antibody (MRK-16) and the P-glycoprotein-MRK-16-Protein A-Sepharose complexes obtained were subjected to a coupled enzyme ATPase assay. 3. While verapamil activated the ATPase, the cyclosporin derivatives inhibited both the substrate-stimulated and the basal P-glycoprotein ATPase. No significant difference was observed between PSC 833 and cyclosporin A on the inhibition of basal P-glycoprotein ATPase activity. PSC 833 was more potent than cyclosporin A for the substrate-stimulated activity. 4. Kinetic analysis indicated a competitive inhibition of verapamil-stimulated ATPase by PSC 833. 5. The binding of 8-azido-[alpha-32P]-ATP to P-glycoprotein was not altered by the cyclosporin derivatives, verapamil, vinblastine and doxorubicin, suggesting that the modulation by these agents of P-glycoprotein ATPase cannot be attributed to an effect on ATP binding to P-glycoprotein. 6. The interaction of the cyclosporin derivatives with ATPase of P-glycoprotein might present an alternative and/or additional mechanism of action for the modulation of P-glycoprotein function.