We tested the hypothesis that the copper present in bile-the major route of elimination of the metal from the body-is derived exclusively from hepatocytes by administering radiocopper (84Cu or 67Cu)-labeled ionic Cu, desialylated (AsCPN) or intact human ceruloplasmin (CPN), intravenously, to rats with cannulated bile ducts. The rates of appearance and the total amounts of radiolabeled isotope recovered in bile were measured. The three vehicles chosen for the delivery of radiocopper interact differently with hepatocytes: ionic Cu is taken up by a passive process (Schmitt, R. C. et al., Am. J. Physiol. 1983; 244:G183-G191); AsCPN is promptly cleared from the circulation by specific receptor-mediated endocytosis, and CPN largely remains in the circulation for the duration of the experiment. Similar amounts of radiocopper were recovered following injections of ionic Cu (3.1%) or CPN (2%), but substantially larger amounts (8.1%) were excreted after administration of AsCPN. Using an antibody to CPN which reacts also with AsCPN, we found about 70% of the bile radioactivity to be immunoprecipitable following injections of either glycoprotein, indicating that a fraction of these copper proteins had entered the bile essentially unmodified. Our observations indicate that in addition to the lysosomal compartment which catabolizes a portion of the AsCPN in hepatocytes, there appears to be a direct route for AsCPN from hepatocellular sinusoids to the bile canaliculi. Since CPN does not interact significantly with hepatocytes, its presence in bile suggests transcytosis via the biliary epithelium.