Freshwater fish Oreochromis niloticus were exposed to Cu in differing Ca2+ levels (15, 30 and 90 mg/L), using acute (0.3 A mu M, 3 d) and chronic (0.03 A mu M, 30 d) exposure protocols and enzyme activities related to the antioxidant (catalase, CAT, EC 220.127.116.11; superoxide dismutase, SOD, EC 18.104.22.168; glutathione peroxidase, GPx, EC 22.214.171.124) and osmoregulation (Total, Na+/K+-ATPase, EC 126.96.36.199, Mg2+-ATPase, EC 188.8.131.52) systems in the erythrocytes were measured. Activities of antioxidant enzymes generally decreased significantly following either Ca2+ alone or Ca2++Cu combinations in both acute and chronic exposures. Na+/K+-ATPase activity significantly decreased in chronic exposures, though there was no clear trend in acute exposures. Mg2+-ATPase activity increased significantly in acute exposures, but not in chronic ones. There were more significant alterations in acute exposure compared to chronic ones. There was no clear trend regarding Cu toxicity and its relationship with Ca2+, which may possibly be prompted by the compensatory mechanisms of the enzymes. It may be concluded that freshwater fish erythrocytes may face different degrees of more physiological stress from different waters.