We provide an extensive characterization of the modulation by p-nitrophenylphosphate, Mg(2+), Na(+), K(+), Rb(+), NH(4)(+) and pH of gill microsomal K(+)-phosphatase activity in the posterior gills of Callinectes ornatus acclimated to low salinity (21 ‰). The synergistic stimulation by K(+) and NH(4)(+) of the K(+)-phosphatase activity is a novel finding, and may constitute a species-specific feature of K(+)/NH(4)(+) interplay that regulates crustacean gill (Na(+), K(+))-ATPase activity. p-Nitrophenylphosphate was hydrolyzed at a maximum rate (V) of 69.2±2.8 nmol Pi min(-1) mg(-1) with K(0.5)= 2.3±0.1 mmol L(-1), obeying cooperative kinetics (n(H)= 1.7). Stimulation by Mg(2+) (V= 70.1±3.0 nmol Pi min(-1) mg(-1), K(0.5)= 0.88±0.04 mmol L(-1)), K(+) (V= 69.6±2.7 nmol Pi min(-1) mg(-1), K(0.5)= 1.60±0.07 mmol L(-1)) and NH(4)(+) (V= 90.8±4.0 nmol Pi min(-1) mg(-1), K(0.5)= 9.2±0.3 mmol L(-1)) all displayed site-site interaction kinetics. In the presence of NH(4)(+), enzyme affinity for K(+) unexpectedly increased by 7-fold, while affinity for NH(4)(+) was 28-fold greater in the presence than absence of K(+). Ouabain partially inhibited K(+)-phosphatase activity (K(I)= 320±14.0 μmol L(-1)), more effectively when NH(4)(+) was present (K(I)= 240±12.0 μmol L(-1)). We propose a model for the synergistic stimulation by K(+) and NH(4)(+) of the K(+)-phosphatase activity of the (Na(+), K(+))-ATPase from C. ornatus posterior gill tissue.