Based on sequence homology to the human organic anion transporting polypeptide 2 (OATP2; SLC21A6), we cloned a new member of the SLC21A superfamily of solute carriers, termed OATP8 (SLC21A8). The protein of 702 amino acids showed an amino acid identity of 80% with human OATP2. Based on Northern blotting, the expression of OATP8 was restricted to human liver. Cosmid clones containing the genes encoding human OATP1 (SLC21A3), OATP2 (SLC21A6), and OATP8 (SLC21A8) served to establish their genomic organization. All three genes contained 14 exons with 13 identical splice sites when transferred to the amino acid sequence. An antibody raised against the carboxyl terminus localized OATP8 to the basolateral membrane of human hepatocytes and the recombinant glycoprotein, expressed in MDCKII cells, to the lateral membrane. Transport properties of OATP8 were studied in stably transfected MDCKII and HEK293 cells. Organic anions transported by human OATP8 included sulfobromophthalein, with a K(m) of 3.3 microm, and 17beta-glucuronosyl estradiol, with a K(m) of 5.4 microm. Several bile salts were not substrates. Thus, human OATP8 is a new uptake transporter in the basolateral hepatocyte membrane with an overlapping but distinct substrate specificity as compared with OATP2, which is localized to the same membrane domain.