Organic anion transporting polypeptides (humans OATPs, rodents Oatps) are expressed in most mammalian tissues and mediate cellular uptake of a wide variety of amphipathic organic compounds such as bile salts, steroid conjugated, oligopeptides and a large list of drugs, probably by acting as anion exchangers. In the present study we aimed to investigate the role of the extracellular pH on the transport activity of 9 human and 4 rat OATPs/Oatps. Furthermore, we aimed to test the concept that OATP/Oatp transport activity is accompanied by extrusion of bicarbonate. By using amphibian Xenopus laevis oocytes expressing OATPs/Oatps and mammalian cell lines stably transfected with OATPs/Oatps, we could demonstrate that in all OATPs/Oatps investigated, with the exception of OATP1C1, a low extracellular pH stimulated transport activity. This stimulation was accompanied by an increased substrate affinity as evidenced by lower apparent Km values. OATP1C1 is lacking a highly conserved histidine in the third transmembrane domain, which was shown by site directed mutagenesis to be critically involved in the pH dependency of OATPs/Oatps. Using online intracellular pH measurements in OATP/Oatp transfected CHO-K1 cells we could demonstrate the presence of a 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid sensitive chloride/bicarbonate exchanger in CHO-K1 cells and that OATP/Oatp mediated substrate transport is paralleled by bicarbonate efflux. We conclude that the pH dependency of OATPs/Oatps may lead to a stimulation of substrate transport in an acidic microenvironment and that the OATP/Oatp mediated substrate transport into cells is generally compensated or accompanied by bicarbonate efflux.