The in vivo interaction of low density lipoproteins (LDL) with the liver was investigated by visualizing the endocytic route using light- and immunoelectron microscopic methods in control and 17 alpha-ethinyl estradiol (EE)-treated rats. The fluorescent dye dioctadecyl indocarbocyanine perchlorate allowed the visualization of LDL at the light microscopic level. Cryoimmunocytochemistry using antibodies against apolipoprotein B was applied at the electron microscopic level. In treated, as well as in EE-treated rats, dioctadecyl indocarbocyanine perchlorate-LDL was taken up by Kupffer cells to a substantial extent. Parenchymal cell uptake was strongly increased after EE treatment and at 10 minutes after injection, LDL was found to be attached to the microvilli of the parenchymal cells and some LDL was already localized in multivesicular structures. At later time points, substantial labeling in multivesicular structures, vesicles near bile canaliculi, and also inside bile canaliculi was observed. A low amount of labeling was found in lysosomes. In untreated rats, label was also observed in the aforementioned structures, but at a much lower level. The biliary appearance of LDL was quantified in rats equipped with permanent catheters in the bile duct, duodenum, and heart. After administration of [125I]tyraminecellobiose-LDL in control rats, about 5% of the injected dose was secreted into the bile during the first 3 hours after injection. This value was about 25% for EE-treated rats. The radioactivity secreted into the bile was trichloroacetic acid-precipitable and high molecular weight bands were immunoreactive for apolipoprotein B as revealed by Western blotting. The described events were not observed when methylated [125I]tyraminecellobiose-LDL was administered. It is concluded that, in rat liver, a significant portion of apolipoprotein B derived from LDL is directly transported to the bile. Since this pathway is enhanced in EE-treated rats, it appears to be a route specific for liver parenchymal cells, dependent on uptake via the LDL receptor.