Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to waterborne Cu (22 µg l^-1) in moderately hard water for up to 28 days. Relative to control fish kept at background Cu levels (2 µg l^-1), Cu pre-exposed fish displayed decreased uptake rates of waterborne Cu via the gills but not of dietary Cu via the gut during 48-h exposures to ⁶⁴Cu radio-labeled water and diet, respectively. At normal dietary and waterborne Cu levels, the uptake rates of dietary Cu into the whole body without the gut were between 0.40 and 0.90 ng g^-1 h^-1, more than 10-fold higher than those of waterborne Cu into the whole body without the gills, which were between 0.02 and 0.07 ng g^-1 h^-1. Previously Cu-exposed fish showed decreased new Cu accumulation in the gills, liver, and carcass during waterborne ⁶⁴Cu exposures and in the liver during dietary ⁶⁴Cu exposures. A 3-h gill Cu-binding assay showed down-regulation of the putative high affinity-low capacity Cu transporters and up-regulation of the low affinity-high capacity Cu transporters on the gills in acclimated fish. Exchangeable Cu pools in all the tissues were higher during dietary ⁶⁴Cu exposures than waterborne ⁶⁴Cu exposures, and previous Cu exposure reduced waterborne exchangeable Cu pools in gill, liver, and carcass. Overall, these results suggest a quantitatively greater role for the dietary route of Cu uptake relative to the waterborne route, a key role for the gill in Cu homeostasis, and important roles for the liver and gut in the normal metabolism of Cu in fish.