Nonacclimated and Cu-acclimated rainbow trout (Oncorhynchus mykiss) exhibited equally rapid clearance of a single bolus of injected ⁶⁴Cu (3,780 nmol/kg) from the plasma (32-40 min to half- concentration). Eight hours after Cu injection, ~80% of the injected Cu was found in the liver. However, when Cu labeled with ⁶⁴Cu was presented intravascularly via continuous infusion at a rate of 158 nmol · kg¹ · h¹ for 72 h, Cu-acclimated fish cleared plasma Cu more effectively than nonacclimated fish. The use of chronically implanted cystic bile duct cannulas revealed a fourfold increase in hepatobiliary Cu excretion in Cu-acclimated fish during infusion, demonstrating the important homeostatic role of the liver in Cu metabolism. Extrahepatobiliary Cu excretion, likely through the gills and apparently exceeding biliary Cu excretion, was evident from appearance of ⁶⁴Cu in the ambient water but was not altered by Cu acclimation. Cu accumulation in white muscle also played an important a role in copper homeostasis.