Detoxification of compounds occurs in two phases. In phase I, a functional group on the toxicant is made available for subsequent Phase II reactions. In phase II, the functional groups are conjugated to a compound that will increase the solubility of the toxicant, enhancing its elimination. Uridine diphosphate glucuronyl transferase (UDPGT) is one microsomal enzyme involved in phase II reactions. It catalyzes the conjugation of toxic compounds with glucuronic acid in reactions in which uridine diphosphate glucuronic acid (UDPGA) is the donor substrate. A new purification procedure for UDPGT has been developed. This procedure includes a Polyethylene glycol fractionation, ion exchange chromatography with DEAE Bio-gel A and affinity chromatography with UDP-hexanolamine-Sepharose. The purification was monitored for three different substrates, bilirubin, 4-nitrophenol (PNP) and 7-hydroxy-4-methylcoumarin (HMC). For this last substrate, HMC, a new continuous fluorometric assay was developed. The purification fold and activity recovery, respectively, towards each substrate was as follows: bilirubin, 31 and 80%; PNP, 31 and 80%; and HMC, 24 and 60%. The significance of these results is discussed with reference to the activation of UDPGT in microsomes by detergents and the reactivation of purified UDPGT by phospholipids. / M.S.