The kinetic properties of the substrate and cation-binding sites of a microsomal gill (Na⁺, K⁺)-ATPase from the hermit crab Clibanarius vittatus were characterized using the synthetic substrate p-nitrophenylphosphate. Substrate was hydrolyzed obeying Michaelis-Menten kinetics at a maximum rate of 47.4 ± 1.9 U mg^-1 with K_M = 1.0 ± 0.04 mmol L^-1. Stimulation of K⁺-phosphatase activity by Mg²⁺ (V_M = 43.9 ± 1.5 U mg^-1 and K_0.5 = 0.6 ± 0.1 mmol L^-1), K⁺ (V_M = 48.9 ± 2.4 U mg^-1 and K_0.5 = 3.7 ± 0.1 mmol L^-1) and NH₄⁺ (V_M = 50.1 ± 1.7 U mg^-1 and K_0.5 = 19.3 ± 0.7 mmol L^-1) showed cooperative kinetics. Stimulation by K⁺ or NH₄⁺ of K⁺-phosphatase activity was similar, enzyme specific activity not varying noticeably in the presence of both ions. The data suggest that after binding of either K⁺ or NH₄⁺ to their sites, increasing concentrations of the other do not displace the first ion from its binding site. Na⁺ (K_I = 16.1 ± 0.2 mmol L^-1), ouabain (K_I = 340.4 ± 16.1 µmol L^-1) and orthovanadate inhibited 70-90% of p-nitrophenylphosphatase activity. This is the first known kinetic characterization of K⁺-phosphatase activity in a hermit crab and provides a useful tool for comparative biochemical studies of (Na⁺, K⁺)-ATPase activities in crustacean gill tissues.