Phenolphthalein is an odorless, tasteless compound, poorly soluble in water, but readily soluble in alcohol or ether. Although its laxative activity was discovered in 1902, the mechanism of action remains unclear. Phenolphthalein has been found to be an ineffective laxative in both jaundiced patients and in experimental animals with ligated common bile ducts. In contrast to the poor solubility of phenolphthalein, phenolphthalein glucuronide is highly water-soluble. In addition, phenolphthalein glucuronide is found in bile following oral phenolphthalein administration, suggesting an enterohepatic circulation. We compared the effect of phenolphthalein and phenolphthalein glucuronide on net water absorption of the ileum and colon of Sprague-Dawley rats using a single-pass perfusion technique. Perfusion of phenolphthalein (10 mg/dl in 154 mM NaCl with 0.5% ethanol and 0.1 g/l gum arabic) resulted in significantly reduced water absorption in both the ileum and colon (66% and 64%, respectively, P less than 0.05). In contrast, phenolphthalein glucuronide did not have a significant effect on net water flux compared to control perfusion in either the ileum or colon. In the ileum only 77 +/- 3% and colon 71 +/- 1.2% of the phenolphthalein infused was recovered, suggesting that much of the infused phenolphthalein was absorbed. In contrast there was no evidence of absorption of phenolphthalein glucuronide, as 98.9 +/- 1.2% was recovered from ileal and colon perfusions. Phenolphthalein glucuronide is not the active form of phenolphthalein, although it may be an effective laxative after deconjugation by colonic bacteria.