A technique has been developed which rapidly separates and purifies UDP-glucuronosyltransferases from liver microsomes of untreated rabbits. by use of this method, highly purified estrone and p-nitrophenol UDP-glucuronosyltransferases can be obtained in good yield in about 48 hr. Microsomes were solubilized with the nonionic detergent Emulgen 911 in low ionic strength buffers and applied to a DEAE-cellulose column equilibrated with low ionic strength buffers. UDP-glucuronosyltransferase activities were then eluted in a stepwise fashion with increasing concentration of KCl. Three fractions were studied. The first two fractions contained only estrone UDP-glucuronosyltransferase activity while a third contained p-nitrophenol UDP-glucuronosyltransferase activity. Each fraction was directly applied to a UDP-hexanolamine Sepharose-4B column, which was then washed extensively with KCl, and the transferases were eluted with UDP-glucuronic acid. A method for separating the transferases on the affinity column is presented. Testosterone and morphine could not be conjugated by any of the purified enzymes.