Three hydroxylated metabolites of methadone, 2-ethyl-3-(dihydroxyphenyl)-5-methyl-3-phenylpyrroline (di-HO-EMDP), 2-ethyl-3-(hydroxymethoxyphenyl)-5-methyl-3-phenylpyrroline (HO-MeO-EMDP) and 2-ethyl-3-(hydroxyphenyl)-5-methyl-3-phenylpyrroline (HO-EMDP) were excreted as O-glucuronide conjugates in bile from the isolated perfused rat liver following the addition of either d- or l-methadone to the perfusate. These glucuronide metabolites were identified as intact molecules by combined gas chromatographic-mass spectrometric analysis of their permethylated derivatives.