A recent 2-year carcinogenicity/toxicology study determined that phenolphthalein (PHTH) is a multisite carcinogen in both mice and rats at all doses evaluated. In response to this finding the metabolism and disposition of PHTH has been evaluated in both F344 rats and B6C3F1 mice at a single oral dose of 800 mg/kg. This dose fell within the range previously found to be carcinogenic in rats and mice. Studies were also performed using 1 and 50 mg/kg doses. At 800 mg/kg recovery of [14C]PHTH after 72 h was near 100% in females but closer to 75% in males. Radioactivity was primarily recovered in the feces in rats (> 90%), while mice excreted 30-40% of administered activity in the urine. There was no significant retention of radioactivity in tissues by 72 h and no significant accumulation of radioactivity in any tissue at any time point. Covalent binding to protein in target tissues, bone marrow and ovary, was at or less than the pmol/mg protein range. The major metabolite was PHTH glucuronide. Three minor metabolites were detected. A sulfate conjugate and and a hydroxylated metabolite were identified by comparison of retention times and 1H NMR and/or mass spectra with synthetic standards. A diglucuronide conjugate was tentatively identified. Biliary elimination was extensive in rats (35% of dose within 6 h); the only product detected in bile was phenolphthalein glucuronide.