This investigation provides an extensive characterization of the modulation by ATP, Mg 2+ , Na + , K + and NH 4 + of a gill microsomal (Na + ,K + )-ATPase from Callinectes danae acclimated to 15‰ salinity. Novel findings are the lack of high-affinity ATP-binding sites and a 10-fold increase in enzyme affinity for K + modulated by NH 4 + , discussed regarding NH 4 + excretion in benthic marine crabs. The (Na + ,K + )-ATPase hydrolyzed ATP at a maximum rate of 298.7±16.7 nmol Pi min −1 mg −1 and K 0.5 =174.2±9.8 mmol L −1 , obeying cooperative kinetics (n H =1.2). Stimulation by sodium (V=308.9±15.7 nmol Pi min −1 mg −1 , K 0.5 =7.8±0.4 mmol L −1 ), magnesium (299.2±14.1 nmol Pi min −1 mg −1 , K 0.5 =767.3±36.1 mmol L −1 ), potassium (300.6±15.3 nmol Pi min −1 mg −1 , K 0.5 =1.6±0.08 mmol L −1 ) and ammonium (V=345.1±19.0 nmol Pi min −1 mg −1 , K 0.5 =6.0±0.3 mmol L −1 ) ions showed site–site interactions. Ouabain inhibited (Na + ,K + )-ATPase activity with K I =45.1±2.5 μmol L −1 , although affinity for the inhibitor increased (K I =22.7±1.1 μmol L −1 ) in 50 mmol L −1 NH 4 + . Inhibition assays using ouabain plus oligomycin or ethacrynic acid suggest mitochondrial F 0 F 1 - and K + -ATPase activities, respectively. Ammonium and potassium ions synergistically stimulated specific activity up to 72%, inferring that these ions bind to different sites on the enzyme molecule, each modulating stimulation by the other.