1. The present study was performed to evaluate and compare the ability of human MDR1-, and rat Mdr1b- and Mdr2-P-glycoproteins to transport hydrophilic monoquaternary drugs. Transport studies were performed with plasma membrane vesicles isolated from MDR1-, Mdr1b-, or Mdr2-overexpressing insect cells. 2. As model substrates we used the N-methylated derivatives of the diastereomers quinidine and quinine, the monoquaternary compounds N-methylquinidine and N-methylquinine. Vincristine, an established MDR1 substrate, was used as a reference. 3. We observed ATP-dependent uptake of all drugs studied into MDR1- and Mdr1b-expressing vesicles. Mdr2 was not able to transport these compounds. MDR1- and Mdr1b-mediated transport was saturable, and could be inhibited by various drugs, including PSC-833. 4. For both MDR1 and Mdr1b the V(max)/K(m) ratios (or clearance) of N-methylquinidine were greater than those determined for N-methylquinine. This stereoselective difference was also evident from differential inhibitory studies with the two isomers. 5. Comparison of normalized clearance indicated that human MDR1 was more effective in transporting the tested substrates than rat Mdr1b. 6. In conclusion, our results demonstrate that MDR1 and Mdr1b, but not Mdr2, are able to transport the monoquaternary model drugs; both MDR1 and Mdr1b display stereospecificity for these cations; and indicate human MDR1 is more efficient in transporting these cations than its rat orthologue Mdr1b.