Methyl 1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) is a novel inhibitor of the ileal apical sodium-dependent bile acid transporter (ASBT/SLC10A2) developed for the treatment of hypercholesterolemia. The present study investigated the hypocholesterolemic action of S-8921 glucuronide (S-8921G) in rats. The plasma concentration of S-8921G was higher than that of S-8921 after single oral administration of S-8921 in normal rats, and S-8921G was excreted into the bile (13% dose). Oral administration of either S-8921 or S-8921G reduced the serum total cholesterol, particularly nonhigh-density lipoprotein cholesterol, in hypercholesterolemic normal rats. In Gunn rats devoid of UDP glucuronosyltransferase-1A activity, S-8921G was undetectable both in the plasma and bile specimens, and only S-8921G administration significantly reduced the serum nonhigh-density lipoprotein cholesterol. An in vitro inhibition study showed that glucuronidation converts S-8921 to a 6000-fold more potent inhibitor of human ASBT (K(i) = 18 nM versus 109 microM). S-8921G was detected both in the portal plasma and loop when S-8921 was administered into the loop of the rat jejunum, although the cumulative amount of S-8921G recovered in the bile was 5-fold greater than that in the loop. The uptake of S-8921G by freshly prepared rat hepatocytes was saturable, and sodium-dependent and -independent systems were involved. Organic anions, such as bromosulfophthalein, estrone 3-sulfate, and taurocholic acid, inhibited the uptake. These results suggest that UDP glucuronosyltransferase-1 isoforms play a critical role in the hypocholesterolemic action of S-8921 by converting S-8921 to a more potent ASBT inhibitor, and organic anion transporter(s) are also involved in its pharmacological action through the biliary excretion of S-8921G.