To examine the substrate specificity of an ATP-dependent organic anion transporter, the multidrug resistance protein 2, we examined the effects of various bile acid conjugates and organic anions on the biliary excretion of phenolphthalein glucuronide, a hydrophilic glucuronide conjugate, in rats. Biliary phenolphthalein glucuronide excretion was markedly inhibited by taurolithocholate-3-sulfate and ursodeoxycholate-3-O-glucuronide. In contrast, ursodeoxycholate-3,7-disulfate and pravastatin only slightly inhibited and cefpiramide did not inhibit biliary phenolphthalein glucuronide excretion. Biliary excretion of sulfobromophthalein, leukotriene C(4) and pravastatin was inhibited by phenolphthalein glucuronide infusion to some extent. These findings suggest that phenolphthalein glucuronide is a relatively low affinity substrate for the multidrug resistance protein 2.