In the presence of 10 microM 2-oxoglutarate (2-OG) and of an inward Na+ gradient, uphill [3H]p-aminohippurate (PAH) uptake occurs due to cooperation of the PAH/2-OG exchanger and the Na(+)-coupled 2-OG transporter in bovine renal basolateral membrane vesicles. Uphill PAH uptake is observed with Cl-, but not with gluconate as the bulk anion. To determine specificity and nature of this anion effect [3H]PAH uptake was measured in the presence of several anions without and with ionophores to distinguish indirect from direct effects on the PAH transporter. Na(+)-gradient plus 2-OG-stimulated [3H]PAH uptake is fast with Cl-, intermediate with F-, Br-, I-, NO3- and SCN-, and slow in the presence of gluconate, SO4(2-) and HPO4(2-). Stimulation by Cl-(as compared to gluconate) is attenuated but not abolished, by clamping electrical potential and pH differences to zero, suggesting a partial effect through charge compensation and a major effect of anions on the PAH transporter itself. Indeed, [3H]PAH/2-OG and [3H]PAH/PAH exchange rates under voltage- and pH-clamped condition depend on bulk anions although the anion effects are less pronounced than with Na(+)-gradient plus 2-OG-stimulated [3H]PAH uptake. Since an inward Cl- gradient does not drive [3H]PAH above or below equilibrium distribution, Cl- ions are most probably not translocated by the PAH transporter. We propose that anions modulate the PAH transporter by interacting with a site not directly related to anion transport.