To protect cells from toxicity, metal-sensitive cellular compartments must be insulated against essential but toxic metals [such as copper (Cu)] accumulated in excess of metabolic requirements. We measured Cu concentrations at the organ and hepatic subcellular levels in juvenile rainbow trout (Oncorhynchus mykiss) during exposure to sublethal waterborne Cu (40 microg/L) for 21 days. There was a time-dependent accumulation of Cu in the gill, liver, plasma, and carcass, with significant difference in Cu-exposed fish relative to the controls being evident by day 7. This significant accumulation of Cu was not associated with impaired growth. Copper concentrations in purity-tested liver subcellular fractions normalized to the liver protein concentration were in decreasing order: organelles > heat-stable proteins > nuclei-debris > NaOH-resistant granules > heat-labile proteins. As a proportion of the total, the majority of the hepatocellular Cu burden (60-68%) was associated with a metabolically active pool (organelles, nuclei-debris, and heat-stable proteins) and the remainder (32-40%) was associated with a metabolically detoxified pool (heat-stable proteins and NaOH-resistant granules) irrespective of the Cu-exposure regime. Because Cu concurrently accumulated in metabolically active and detoxified pools, we conclude that the spillover hypothesis of metal toxicity did not hold under the exposure conditions employed in this study. Moreover, these data suggest that rainbow trout can withstand significant above-background Cu accumulation in hepatic putative metal-sensitive compartments without chronic toxic effects at the organism level.