In an earlier publication from this laboratory (1) the separation of monoglucosiduronate, monosulfate, sulfoglucosiduronate, and disulfate conjugates of estrone (E,)3 and/or 17/3-estradiol (E,) was described. The technique involved elution of DEAE-Sephadex (A 25) columns with simple concentration gradients of NaCl in H,O and was based on the original studies of Hahnel (2). It was also pointed out (1) that a certain degree of separation of estrone-3-glucosiduronate (E,3G) and 17/3estradiol-3-glucosiduronate (E,3G) was possible, while the latter compound could be partially separated, in turn, from 17p-estradiol-17glucosiduronate (Ez17G). More recently, continued experience with the above method has resulted in practically complete separation of those three monoglucosiduronates from each other. This procedure has proved to be of particular value in the study of estrogen glucosiduronate metabolism in the human (3, 4). The present report concerns the exact methodology now being applied in our laboratory and the results which may be expected from it. MATERIALS 17/3-Estradiol-6,7-3H-17-glucosiduronate, specific activity (SA) = ca. 1 mCi/pmole, was purchased from New England Nuclear Corp., Boston, Mass., and was purified as described elsewhere (5). The 3-gluco-