The organic cation transporter, OCT2, plays a role in renal secretion of a broad array of weak bases. To determine whether the degree of ionization of these compounds plays a role in their interaction with OCT2, we examined the influence of external pH values on the activity of the human ortholog of OCT2, as expressed in Chinese hamster ovary-K1 cells. Importantly, changing the pH value from 7.0 to 8.0 had no effect on the rate of transport of the fixed cations, tetraethylammonium and 1-methyl-4-phenylpyridinium, i.e. the pH value did not have an effect upon the transporter itself. Cimetidine (pK(a) 6.92), a competitive inhibitor of hOCT2, displayed a 3.5-fold increase in IC(50) as pH values increased from 7 to 8. hOCT2-mediated cimetidine transport decreased over this pH range, the consequence of an increase in K(t) and decrease in J(max) at the higher pH value. The weak bases trimethoprim and 4-phenylpyridine showed a similar pattern of pH-sensitive interaction with hOCT2. The non-ionizable sterol, corticosterone, also inhibited hOCT2 activity, although it was neither competitive in nature nor was it sensitive to pH in the manner observed with weak bases. We conclude that the degree of ionization plays a critical role in binding of substrate to organic cation transporters.