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Notes: Hepatocytes were isolated by established procedures from freshly-excised livers of ovariectomized rats. Integrity of the cells was verified by DNA, protein, and calcium contents, and by dye exclusion. The cells also showed progressive increments in oxidation to 14CO2 of [26-14C]cholesterol during one to five hours' incubation. Analysis was undertaken of cellular reactivities toward estrogen and the hepatocarcinogen dibutylnitrosamine (DBN). Binding and retention of [3H]estradiol-17β (E2β) by isolated liver cells was specific for E2β, saturable, temperature-dependent, and maximal after 30-minute incubation. The apparent dissociation constant for the binding process at 22°C is 2 × 10-9 M, and the total number of binding-sites at saturation corresponds to approximately 3,400 E2β molecules per liver cell. To probe for steroid binding-sites at their external surfaces, cells were incubated 30 minutes with mounted 17β-estradiol-17-hemisuccinyl:albumin:nylon fibers. The covalentlyimmobilized estrogen (1 ng/mg albumin) was accessible for interaction with antiserum directed against 17β-estradiol-17-hemisuccinyl:albumin. Significant numbers of isolated liver cells were retained by estrogen-derivatized fibers at 22°C after extensive washes. Binding was markedly reduced by incubation at 4°C and by prior exposure to free E2β (× 10-8 M), but not to the relatively inert estradiol-17α (E2α). Fiber-bound cells could be dislodged by brief incubation in 150 mOsM saline with 2 × 10-7 M E2β or diethylstilbestrol, but not E2α, cortisol, progesterone, or testosterone, and recovered intact. Cells that had been retained by the fibers and those that were not adherent were collected and washed under identical conditions, then plated in serum-free, chemically-defined medium at 37°C. After 72 hours, specific binding of E2β by the fiber-binding cells during 30 minutes' incubation was 2.5-fold that of cells which had not bound the immobilized steroid. Similarly, stimulation of the oxidation to 14CO2 of [26-14C]cholesterol by E2β was greater in fiber-binding than in non-binding liver cells after three hours' incubation. In the absence of added mitogen, thymidine incorporation into macromolecular form (20 hours), and cell proliferation (48 hours) were significantly greater in fiber-binding cells as compared to non-binding hepatocytes. Moreover, in parallel experiments, when cells were exposed to 1 × 10-9 M estrogens or to 1 × 10-4 M nitrosamines to assess the capacities of these substances to increase basal thymidine incorporation, total DNA, and cell numbers, only those cells with estrogen-binding sites at their surfaces showed significant E2β- and DBN-induced increments in these parameters as compared with paired controls that had been treated with E2α or the noncarcinogen diphenylnitrosamine. These data indicate that the accessibility of hormone-binding components at the plasma membrane may contribute to the capacity of a given liver cell to respond to E2β, as well as to other known hepatocarcinogens.