Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to waterborne Cu (22 µg/l) in moderately hard water for up to 28 days. Relative to control fish kept at background Cu levels (2 µg/l), Cu-preexposed 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-radiolabeled 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 0.40-0.90 ng · g¹ · h¹, >10-fold higher than uptake rates of waterborne Cu into the whole body without the gills, which were 0.02-0.07 ng · g¹ · h¹. 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 downregulation of the putative high-affinity, low-capacity Cu transporters and upregulation of the low-affinity, high-capacity Cu transporters at the gills in Cu-preexposed fish. Exchangeable Cu pools in all the tissues were higher during dietary than during 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 than for the waterborne route of Cu uptake, 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.