P-glycoprotein (P-gp) is a transmembrane glycoprotein that confers multidrug resistance (MDR). It has been demonstrated that the Gly185 residue within the cytoplasmic loop between predicted transmembrane portions 2 and 3 plays an important role in substrate specificity of human P-gp. Derivatives of cyclosporin interact with and reverse the ability of P-gp to act as a drug efflux pump. To determine if the Gly185 residue of human P-gp is also important for the interaction of P-gp with closely related cyclosporin derivatives, we examined the effect of PSC-833 and CsA on P-gp in KB3-1 cells transfected with human wild-type P-gp (GSV-2) or with the mutant P-gp (VSV-1) that habored the Gly185-->Val substitution. While the ability of CsA to sensitize VSV-1 cells to anticancer agents was enhanced, no changes in the potency of PSC-833 against cells transfected with either the wild-type or mutant P-gp were observed. In addition, VSV-1 transfected cells were more sensitive to CsA inhibition of verapamil-stimulated ATPase activity than cells transfected with wild-type P-gp. Furthermore, the intracellular accumulation of CsA was low in GSV-2 P-gp-expressing cells, compared with its accumulation in VSV-1 cells and it was found to be as high as in non-P-gp expressing KB3-1 cells. These results indicated an enhanced sensitivity of Val185-P-gp expressing cells to CsA that correlated with increased intracellular accumulation in these cells. In contrast, no significant difference in the accumulation of PSC-833 was observed among the parental, wild-type or resistant cells. Since PSC-833 was found to be more potent than CsA, these studies provided insight into the effects of the structure of MDR modulators in mediating sensitivity to anticancer drugs.