The gill (Na⁺, K⁺)-ATPase plays a pivotal role in the osmoregulatory processes of aquatic crustaceans. A systematic and comparative study of the biochemical characteristics of gill enzymes from crustaceans adapted to different habitats should allow a better understanding of the biochemical adaptations that have lead to the colonization of the estuarine and freshwater habitats. Adult, intermolt C. ornatus were collected from Ubatuba bay (São Paulo State, Brazil), and (Na⁺, K⁺)-ATPase-rich microsomes were prepared from the posterior gills according to Furriel et al. (J. Exp. Zool. 301A: 63, 2004). K⁺-phosphatase activity was assayed continuously at 25 °C in 50 mM Hepes buffer, pH 7.5 containing 10 mM p-nitrophenylphosphate (PNPP), 7 mM MgCl₂ and 10 mM KCl. Sucrose density gradient centrifugation of the microsomal fraction revealed a single protein peak, coincident with the (Na⁺, K⁺)-ATPase activity. Stimulation of K⁺-phosphatase activity by PNPP revealed site-site interactions (n= 1.6), with a maximal rate of V= 52.0 ± 2.6 U/mg and K_0.5= 1.1 ± 0.1 mM. Magnesium (V= 52.8 ± 2.6 U/mg; K_0.5 = 1.0 ± 0.1 mM) and K⁺ ions (V= 47.2 ± 2.3 U/mg; K_0.5= 2.3 ± 0.2 mM) also stimulated PNPP hydrolysis obeying cooperative kinetics. Western blot analysis revealed a single, immunoreactive band coincident with a 100-kDa protein band, suggesting the presence of a single a-subunit isoform in C. ornatus gill tissue. Sodium ions (K_I= 1.7 ± 0.1 mM) and ouabain (K_I= 201.4 ± 10.2 mM) inhibited the K⁺-phosphatase activity by 90 and 76%, respectively. The kinetic properties of the (Na⁺, K⁺)-ATPase from C. ornatus are very similar to those for C. danae, an estuarine crab.