Branchial Na,K-ATPase activity was characterized in 3 freshwater fish species (Oncorhynchus mykiss, Oreochromis niloticus, and Cyprinus carpio) with different ecological needs. Na+, K+, and Cl- concentrations in the gills were also measured. The maximal Na,K-ATPase activity was observed at 100 mM Na+, 20-40 mM K+, 3-4 mM Mg2+, and 1 mM ouabain. The maximal velocity (Vmax) of Na,K-ATPase isolated from O. mykiss (1.07 mu mol Pi/mg prot/h) was lower than that isolated from O. niloticus (7.25 mu mol Pi/mg prot/h) and C. carpio (7.44 mu mol Pi/mg prot/h). Nevertheless, Na+, K+, and Cl- levels in O. mykiss were higher than the others. The lowest Vmax value can be attributed to the highest ion concentrations in the gills of O. mykiss. However, substrate concentrations, which give half of Vmax (Km) of Na,K-ATPase in the gills of O. niloticus and C. carpio, were different although they exhibited similar Vmax values. The low Km value in the gills of O. niloticus compared to C. carpio may be related to high resistance to changing environmental factors.