Fathead minnows exposed to sublethal Cu concentrations may undergo branchial damage followed by repair, and may also develop enhanced Cu tolerance. The primary objective of this study was to determine whether the cycle of damage and repair was necessary for the development of enhanced Cu tolerance. Inferences regarding damage and repair were made from changes in the whole body Na + of juvenile (0.5 g) fathead minnows (Pimephales promelas). Two experiments were conducted in which juvenile minnows were exposed to various sublethal Cu concentrations for 16 d. In the first experiment, fish were exposed to one of four different Cu concentrations (0, 73, 118, 189 μg/L Cu) then challenged with an 8-d exposure at 302 μg Cu/L. Only the fish exposed to the highest Cu dose experienced enhanced Cu tolerance relative to the other three doses. In the second experiment, whole body Na + was monitored in fish exposed to one of five different Cu exposures (0, 70, 127, 202, 289 μg/L Cu). Fish exposed to 70 μg/L Cu did not experience a significant decline in whole body Na + at any point during the 16-d exposure period. The reduction in whole body Na + was short lived and moderate (17%) in the fish exposed to 127 μg/L Cu, but more severe (>30%) and longer lasting in the fish exposed to 202 μg/L Cu. At 289 μg/L Cu, the fish experienced irreversible reductions in whole body Na + and ultimately died. When taken together, results from these two experiments suggest that enhanced tolerance will only develop in fathead minnows that have experienced a pronounced, relatively long-term cycle of branchial damage and repair.