The specific binding of [3H]estradiol-17 beta-(beta-D-glucuronide) ([3H]E217G) was examined in isolated basolateral (bLPM) and canalicular (cLPM) liver plasma membranes. Two distinct binding sites were identified in each membrane fraction by competition and saturation experiments. Binding parameters obtained from competition studies were: Kd1 = 26 nM, Bmax1 = 0.26 pmol/mg protein; Kd2 = 2.6 microM, Bmax2 = 27 pmol/mg protein for bLPM; and Kd1 = 81 nM, Bmax1 = 0.61 pmol/mg protein; Kd2 = 6.7 microM, Bmax2 = 79 pmol/mg protein for cLPM. Binding parameters obtained from saturation experiments were not significantly different. There was no Na+ requirement for binding. Kinetic dissociation experiments showed that binding was reversible and revealed two components. The dissociation rate constants did not vary with the method of dilution of radioligand, i.e. by "infinite" volume, or excess unlabeled ligand, thus ruling out the possibility of cooperativity. The ability of a series of compounds to inhibit the binding of [3H]E217G was also examined. In bLPM, taurocholate (TC), estrone sulfate (E1SO4) and bromosulfophthalein (BSP) were able to compete with both binding sites, whereas estriol-17 beta-(beta-D-glucuronide) (E317G), estriol-16 alpha-(beta-D-glucuronide) (E316G), testosterone glucuronide (TG), estradiol-3-(beta-D-glucuronide) (E23G), estriol-3-(beta-D-glucuronide) (E(3)3G), cholate and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) were able to inhibit binding to only the low-affinity site. In cLPM, only the cholestatic steroid D-ring glucuronides (E(3)17G, E(3)16G and TG) and TC were able to compete with both sites, whereas the non-cholestatic steroid A-ring glucuronides (E(2)3G and E(3)3G), BSP and DIDS competed for only the low-affinity site. Based on the observed substrate specificities, the low-affinity sites in bLPM and cLPM are postulated to represent multispecific organic anion carriers. The high-affinity site in cLPM may play a role in mediating steroid D-ring glucuronide-induced cholestasis.