Gill (Na⁺,K⁺)-ATPase plays a main role in osmo-ionic regulatory processes in hyperosmoregulating crustaceans. The kinetic properties of the enzyme from posterior gills microsomes of the swimming crab Callinectes danae acclimated to 33‰ salinity for 10 days were studied. K⁺-phosphatase activity was assayed continuously, at 25^oC, in 50 mM Hepes buffer, pH 7.5, containing 10 mM p-nitrophenylphosphate (PNPP), 7 mM MgCl₂ and 15 mM KCl, according to Masui et al. (J. Exp. Zool. 303A: 294-307, 2005). The K⁺-phosphatase activity in the gill tissue of 33‰ salinity acclimated crabs (V= 52.8 ± 3.1 U/mg) was 2-fold lower compared to that observed for crabs acclimated to 15‰ salinity (V= 102.9 ± 4.3 U/mg). The enzyme hydrolyzed PNPP obeying cooperative kinetics, with V= 52.8 ± 3.1 U/mg and K_0.5= 1.7 ± 0.1 mM. Magnesium (V= 49.8 ± 2.1 U/mg; K_0.5= 1.1 ± 0.04 mM), K⁺ (V= 55.2 ± 3.2 U/mg; K_0.5= 2.9 ± 0.2 mM) and NH₄⁺ ions (V= 50.5 ± 2.8 U/mg; K_0.5= 14.4 ± 0.8 mM) also stimulated K⁺-phosphatase activity following cooperative kinetics, suggesting the presence of multiple binding sites in the enzyme molecule. As observed for 15‰ salinity acclimated crabs, K⁺-phosphatase activity was not synergistically stimulated by K⁺ and NH₄⁺. Ouabain (K_I= 234.5 ± 11.3 mM) inhibited about 72.3% of total PNPPase activity, indicating the presence of other PNPP hydrolyzing enzymes in the microsomal fraction studied.