The present study compares the uptake and metabolism of 17 beta-estradiol, 17 beta-estradiol 3-glucoside, and 17 beta-estradiol 3-glucuronide by a highly purified preparation of rabbit liver nuclei. The uptake of the three estrogens was rapid and equilibration was reached within 60 s. The order of uptake was 17 beta-estradiol (64 fmol X mg protein -1) greater than 17 beta-estradiol 3-glucoside (10 fmol X mg protein -1) greater than 17 beta-estradiol 3-glucuronide (6.5 fmol X mg protein -1). Thin-layer chromatography of the estrogens taken up by rabbit liver nuclei indicated the presence of a beta-glucosidase activity associated with the nuclear preparation. The apparent Km value of this enzyme for 17 beta-estradiol 3-glucoside (3.5 microM) was about 10-fold higher when compared with the cytosolic enzyme. The uptake of the three estrogens was linearly proportional to the substrate concentration from 1 to 100 nM. No competition for uptake was observed among the steroids and the presence of diethylstilbestrol did not reduce the uptake of the steroids. These findings suggest that 17 beta-estradiol, 17 beta-estradiol 3-glucoside, and 17 beta-estradiol 3-glucuronide are taken up by nuclei by a nonsaturable diffusion process. The effect of cytosol on the uptake of estrogens by purified nuclei was also investigated. It was observed that cytosol reduced the uptake o 17 beta-estradiol but had little effect on that of its conjugates.