The aglycone, 3-hydroxybenzo[ a]pyrene, was metabolized to 3-benzo[ a]pyrenyl-β- d-glucopyranosiduronic acid in the presence of uridine 5′-diphosphoglucuronic acid and rabbit liver microsomes. The course of the biosynthetic reaction was followed by fluorimetry and reverse-phase, paired-ion high pressure liquid chromatography (HPLC). Also, the HPLC system was used to analyze for glucuronide and 3-hydroxybenzo[ a]pyrene during the isolation procedure. The existence of a glucuronide of 3-hydroxybenzo[ a]pyrene was determined by radiotracer and enzymic techniques, utilizing the HPLC system. Field desorption and direct inlet mass spectral techniques were used to characterize the 3-hydroxybenzo[ a]pyrene glucuronide.