1. Gall-bladder bile was obtained at cholecystectomy from seven patients who had been given [64Cu]cupric acetate intravenously.
2. Studies using dialysis and Sephadex gel filtration indicated that most of the 64Cu in the bile was in a heat-stable complex of molecular weight exceeding 50 000. The remaining biliary 64Cu was in a low-molecular-weight component.
3. Above pH 4, 64Cu could not readily be removed from the macromolecular complex either by excess carrier copper ions, by d-penicillamine or by EDTA.
4. Absorption of 64Cu from 64Cu-labelled bile injected intraduodenally into rats was significantly less than from [64Cu]cupric acetate similarly injected.
5. These observations suggest that limitation of an entero-hepatic circulation of copper by a biliary macromolecule which differs from caeruloplasmin, may be a significant factor in copper homeostasis in man.