Both the osmoregulatory and excretory processes in aquatic crustaceans take place mainly across the gill epithelium. The gill Na⁺,K⁺-ATPase is a key enzyme in osmoregulatory processes, and a putative role for this enzyme in ammonia excretion has been recently proposed. We examined the effect of NH₄⁺ on Na⁺,K⁺-ATPase activity in a gill microsomal fraction from M. amazonicum. Adult intermolt shrimps were collected from Usina São Geraldo lagoon (Sertãozinho-SP) and Na⁺,K⁺-ATPase-rich microsomes were obtained according to Furriel et al. (Comp. Biochem. Physiol. 126B: 303, 2000). Na⁺,K⁺-ATPase activity was assayed continuously, at 25°C; standard assay conditions were 2 mM ATP, 3 mM MgCl₂ and 40 mM NaCl in 50 mM Hepes buffer, pH 7.5. Under saturating concentrations of ATP, Na⁺ and Mg²⁺, the stimulation of ATP hydrolysis by K⁺ (V= 271.8 ± 10.9 U/mg; K_M= 0.88 ± 0.04 mM) and NH₄⁺ (V= 385.9 ± 15.8 U/mg; K_M= 5.02 ± 0.21 mM) followed Michaelian kinetics. In the presence of fixed NH₄⁺ concentrations (2–50 mM), K⁺ ions also stimulated ATPase activity through Michaelian kinetics, resulting in increasing specific activities up to 362.4 ± 27.2 U/mg, with K_M values raising up to 1.90 ± 0.14 mM. In contrast, the presence of fixed K⁺ concentrations (1-10 mM) did not affect the kinetic parameters obtained for the stimulation of enzyme activity by NH₄⁺. Under saturating concentrations of ATP, Mg²⁺, Na⁺ and K⁺, total ATPase activity was inhibited by ouabain (3 mM) to similar residual values (~ 50 U/mg) both in the presence (K_I= 35.6 ± 2.1 mM) and absence (K_I= 147.5 ± 8.9 mM) of 50 mM NH₄⁺, confirming that the Na⁺,K⁺-ATPase is in effect responsible for the synergistic stimulation of ATPase activity by K⁺ and NH₄⁺. The results strongly suggest the existence of two different sites that may be occupied by K⁺ and/or NH₄⁺ in the enzyme from M. amazonicum gill tissue, what may be of physiological relevance to osmoregulatory and excretory processes in palaemonid shrimps.