The kinetic properties of a microsomal gill (Na⁺, K⁺)-ATPase from juvenile and adult Amazon River shrimp Macrobrachium amazonicum were characterized using the synthetic substrate p-nitrophenylphosphate. The substrate was hydrolyzed revealing cooperative kinetics at maximum rates of 71.3 ± 1.1 U mg^-1 and 82.7 ± 2.3 U mg^-1 with K_0.5 = 1.45 ± 0.02 mmol L^-1 and 1.52 ± 0.04 mmol L^-1 for juveniles and adults, respectively. Stimulation of K⁺-phosphatase activity by Mg²⁺ and K⁺ also exhibited cooperative kinetics independently of ontogenetic stage. While the K_0.5 values estimated for Mg²⁺ and K⁺ stimulation of K⁺-phosphatase activity were very similar, maximum rates for adult shrimps were ≈1.8-fold greater than that for juveniles. NH₄⁺ stimulation resulted in a ≈10-fold increase in K_0.5 for both stages compared to Mg²⁺ and K⁺. Further, when stimulated by NH₄⁺ plus K⁺, p-nitrophenylphosphate hydrolysis was mostly unchanged, suggesting that NH₄⁺ and K⁺ bind to the same site. While K_0.5 values varied, stimulation by NH₄⁺ plus K⁺ was not synergistic. Increasing NH₄⁺ or K⁺ concentrations do not displace the corresponding ion from the cation-binding site. Ouabain partially inhibited p-nitrophenylphosphatase activity (K_I = 142.0 ± 5.6 µmol L^-1 and 156.0 ± 6.2 µmol L^-1 for juveniles and adults, respectively); however, inhibition was more effective when NH₄⁺ was present (K_I = 117.1 ± 4.6 µmol L^-1 and 47.2 ± 1.9 µmol L^-1 for juveniles and adults, respectively). Although the activity of ATPases other than the (Na⁺, K⁺)-ATPase are considerably greater in adult shrimps, the kinetic properties of the gill K⁺-phosphatase activity in the two life cycle stages, and the affinities for p-nitrophenylphosphate and ions are similar.