Multidrug resistance protein Mrp2 (symbol Abcc2) in liver plays a significant role in the biliary excretion of organic anionic conjugates. Mutations in human MRP2 result in defects in excretion of conjugated bilirubin and other cholephiles known as the Dubin-Johnson syndrome. Previous studies indicate that transporters with Mrp2-like functions are present in ancient vertebrates. We have now characterized an Mrp2 ortholog at the molecular level from the liver of the small skate, Raja erinacea, a marine vertebrate that evolved ~200 million years ago. The full-length skate Mrp2 cDNA is 6 kb and encodes for a 1,564-amino acid peptide with 56% identity to human Mrp2. Northern blot analysis demonstrated that skate Mrp2 is abundantly expressed in skate liver, intestine, and kidney. Immunoblots reveal a 180-kDa protein in skate liver. Immunofluorescence studies locate skate Mrp2 to the apical membrane of hepatocytes, renal tubules, and intestine. A PDZ-interacting motif is also found at its COOH terminus. Further sequence analysis indicates that transmembrane domains 1, 9, 11, 16, and 17 are the most highly conserved transmembrane domains between skate Mrp2 and mammalian MRP2/Mrp2s. This analysis indicates that Mrp2 orthologs evolved early in vertebrate evolution and that conserved domains may be important determinants of Mrp2 substrate specificity.