Uptake of organic solutes and xenobiotics by mammalian cells is mediated by ATP-independent transporters, and four families of transporters have now been identified. To search for novel organic solute transporters, a liver cDNA library from an evolutionarily primitive marine vertebrate, the little skate Raja erinacea, was screened for taurocholate transport activity by using Xenopus laevis oocytes. In contrast to the organic anion transporters identified to date, a transport activity was identified in this library that required the coexpression of two distinct gene products, termed organic solute transporter ␣ and  (Ost␣, Ost). Ost␣ cDNA encodes for a protein of 352 aa and seven putative transmembrane (TM) domains. Ost contains 182 aa and has at least one and perhaps two TM domains. There is no significant sequence identity between Ost␣ and Ost, and only low identity with sequences in the databases; however, Ost␣ bears a resemblance to some G protein-coupled receptors, and Ost exhibits 22% amino acid identity with the C-terminal TM and intracellular domains of protocadherin-␥, a cell surface glycoprotein. Xenopus oocytes injected with the cRNA for both Ost␣ and Ost, but not each separately, were able to take up taurocholate, estrone sulfate, digoxin, and prostaglandin E 2, but not p-aminohippurate or Sdinitrophenyl glutathione. Transport was sodium-independent, saturable, and inhibited by organic anions and steroids, including the major skate bile salt, scymnol sulfate. These results identify an organic anion transporter composed of a putative seven-helix TM protein and an ancillary membrane polypeptide. E pithelial cells continuously extract large amounts of organic solutes, drugs, and other xenobiotics from circulating blood plasma. Some of the transporters responsible for this drug clearance recently have been characterized at the molecular level and include four families of ATP-independent transporters: the Na ϩ -coupled bile acid transporters (NTCPs), the Na ϩ -independent organic anion transporting polypeptides (OATPs), the organic anion transporters (OATs), and the organic cation transporters (OCTs) (1-5). The first member of each of these families was identified by expression cloning in Xenopus laevis oocytes, and additional members subsequently have been identified by homology screening. All of these transporters consist of single polypeptides, which, when expressed in heterologous systems, are able to mediate organic solute transport.Because of the large number of endogenous and exogenous compounds that must be transported by the liver, kidney, intestine, and other tissues, it is likely that other transporters and transporter families also exist, but have not yet been described (5). In an attempt to identify novel organic anion transport proteins we used a comparative approach, screening a liver cDNA library from an evolutionarily ancient vertebrate species, the little skate Raja erinacea. This elasmobranch is thought to have evolved 200 million years ago, yet displays many physiological features...