Na⁺,K⁺-ATPase is involved in active Na⁺ capture through the gill epithelium of crustaceans subjected to low salinities, and freshwater animals acclimated to brackish water usually show a notable decrease in the activity of this enzyme. We have recently shown that the 2-fold lower gill K⁺-phosphatase specific activity observed for the freshwater shrimp M. olfersii acclimated to 21‰ salinity, compared to animals held in fresh water, may derive from expression of a different isoenzyme and not from decreased enzyme expression (Mendonça et al., Comp. Biochem. Physiol., 2006, in press). Here we report a comparative kinetic characterization of the K⁺-phosphatase activity of the enzyme from M. amazonicum acclimated to fresh water or 21‰ salinity. Adult intermolt shrimps were collected from Usina São Geraldo lagoon (Sertãozinho-SP), and maintained for 10 days in fresh water or at 21‰ salinity. Na⁺,K⁺-ATPase-rich microsomes were obtained according to Furriel et al. (J. Exp. Zool. 301A:63, 2004) and K⁺-phosphatase activity was assayed continuously, at 25°C, in 50 mM Hepes buffer, pH 7.5. Western blotting analysis revealed similar a-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water. For high-salinity acclimated shrimps, PNPP hydrolysis occurred with site-site interactions (n= 1.4) with a maximal rate V= 158.1 U/mg and K_0.5= 1.27 mM. Magnesium (V=162.1 U/mg; K_0.5= 1.3 mM), K⁺ (V=159.9 U/mg; K_0.5 = 1.7 mM), and NH₄⁺ ions (V=167.4 U/mg; K_0.5= 12.3 mM) also stimulated substrate hydrolysis obeying cooperative kinetics, and ouabain (K_I= 291.8 mM) inhibited total PNPPase activity by 90%. Interestingly, a similar specific K⁺-phosphatase activity (V= 156.0 U/mg) was determined for animals held in freshwater, and the K_0.5 values for enzyme stimulation by Mg²⁺, K⁺ and NH₄⁺ were not significantly different from those determined for high-salinity acclimated shrimps. In contrast, ouabain completely inhibited PNPPase activity in gill microsomes from freshwater acclimated animals, with a 1.7-fold lower K_I value. The results suggest that, similarly to M. olfersii, M. amazonicum may express different isoenzymes in response to salinity changes, despite the surprisingly similar specific activities found in animals acclimated to fresh or brackish water.