The human liver-specific organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are involved in the elimination of numerous xenobiotics and drugs. Although dogs are frequently used for toxicologic and pharmacokinetic characterization of novel drugs, nothing is known about their OATP1B1/1B3 ortholog. Therefore, we cloned and characterized the first canine organic anion transporting polypeptide from dog liver, termed Oatp1b4. The isolated Oatp1b4 cDNA comprises 3661 base pairs (bp) with an open reading frame of 2076bp, encoding a 692-amino acid protein with a molecular mass of approximately 85kDa. The Oatp1b4 gene is approximately 61kb long and has a similar organization as the human OATP1B1 and OATP1B3 with 13 exons identical in length. Northern blot analysis shows that Oatp1b4 is predominantly expressed in the liver. Oatp1b4 mediates sodium-independent transport of typical organic anions including bromosulfophthalein (BSP), [D-penicillamine(2,5)]enkephalin (DPDPE), estradiol-17beta-glucuronide (E17betaG), estrone-3-sulfate and taurocholate. In addition, Oatp1b4 transports the OATP1B3-specific substrate cholecystokinin octapeptide (CCK-8). Kinetic studies showed that Oatp1b4-mediated E17betaG and estrone-3-sulfate transports were monophasic with K(m) values of 5+/-1microM and 33+/-4microM, respectively. In conclusion, the cloned canine Oatp1b4 will provide additional molecular basis to further characterize the species difference of the OATP1B family members.