An increase in salinity of freshwater can affect the physiology and metal uptake in fish. In the present study, Nile tilapia Oreochromis niloticus were exposed to copper (1.0 mg/l) in increased salinities (2, 4, and 8 ppt) for 0, 1, 3, 7, and 14 days. Following the exposures, the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, and Ca(2+)-ATPase were measured in the gill, kidney, and intestine to evaluate the changes in osmoregulation of fish. Results showed that increases in salinity and Cu exposure of fish significantly altered the ATPase activities depending on the tissue type, salinity increase, and exposure durations. Salinity-alone exposures increased Na(+)/K(+)-ATPase activity and decreased Ca(2+)-ATPase activity. Na(+)/K(+)-ATPase activity decreased following Cu exposure in 2 and 4 ppt salinities, though the activity increased in 8 ppt salinity. Ca(2+)-ATPase activity decreased in the gill and intestine in all salinities, while the activity mostly increased in the kidney. However, there were great variations in Mg(2+)-ATPase activity following exposure to salinity alone and salinity+Cu combination. Cu accumulated in the gill and intestine following 14 days exposure and accumulation was negatively correlated with salinity increase. Data indicated that ATPases were highly sensitive to increases in salinity and Cu and might be a useful biomarker in ecotoxicological studies. However, data from salinity increased freshwaters should carefully be handled to see a clear picture on the effects of metals, as salinity affects both metal speciation and fish osmoregulation.