Human organic anion transporter 4 (hOAT4) is localized at the apical side of proximal tubule cells, facilitating the uptake of estrone sulfate (ES) and dehydroepiandrosterone-sulfate (DHEAS). To date, the transport mode and driving forces of hOAT4 are controversial. To address this question, we performed transport studies using HEK293 cells stably transfected with hOAT4 and showed a Km of 253 mM for 6-carboxyfluorescein (6-CF). Accumulation of 6-CF and [3H]ES were strongly inhibited by ES, DHEAS, and probenecid, whereas p-aminohippurate (PAH), glutarate exhibited no reduction of hOAT4 transport rate. Urate showed a significant inhibition of [3H]ES uptake with an IC50 value of 1.03 mM and hOAT4 mediated [14C]urate uptake was 40% higher than that into not transfected mock cells. Trans-stimulation studies revealed a significant increase of 6-CF as well as [3H]ES uptake by PAH and glutarate, documenting that hOAT4 works in a asymmetrical mode. [14C]urate and [3H]ES uptake were enhanced by chloride free medium. An acidification of the external medium stimulate 6-CF and ES uptake, suggesting organic anion/ Cl^-or OH^-exchange. Taken together, human OAT4 can exchange extracellular organic anions (estrone sulfate or urate, but not PAH and glutarate) against intracellular inorganic anions (Cl^? or ???^-)?and organic anions (PAH, glutarate, and, most likely, a- ketoglutarate).