Juvenile rainbow trout were fed diets containing control (0.26 mmol/g) or elevated (1.3 mmol/g) dietary Na+ in combination with either background (19 nmol/L) or moderately elevated levels (55 or 118 nmol/L) of waterborne Cu for 21 d. Unidirectional waterborne Na+ uptake rates (measured with 22Na) were up to four orders of magnitude higher than those of Cu (measured with 64Cu). Chronic exposure to elevated dietary Na+ alone or in combination with elevated waterborne Cu decreased whole-body uptake rates of waterborne Na+ and Cu. Accumulation of new Cu and Na+ at the gills was positively and highly significantly correlated and responded to the experimental treatments in a similar fashion, suggesting that Na+ and Cu have common branchial uptake pathways and that dietary Na+ preexposure modifies these pathways. Chronic exposure to elevated waterborne Cu significantly increased Cu concentrations in the liver but caused only modest increases in total Cu concentrations in the whole body and gill. Chronic exposure to elevated dietary Na+ slightly decreased whole-body Cu concentration on day 14 and greatly reduced liver Cu concentration on days 14 and 21; new Cu accumulation in whole-body, gill, and internal organs was reduced on all days. Chronic exposure to elevated waterborne Cu or dietary Na+ alone reduced short-term gill Cu binding at low waterborne Cu concentrations. At high waterborne Cu concentrations, chronic exposure to elevated waterborne Cu had no effect, while elevated dietary Na+ increased Cu binding to the gills. Combined chronic exposure to elevated dietary Na+ and waterborne Cu decreased gill Cu binding over the entire range of Cu concentrations tested. Clearly, chronic exposure to elevated dietary Na+ and waterborne Cu appears to modify gill Cu-binding characteristics and may be important considerations in future development of a chronic biotic ligand model for Cu.