We characterize the kinetic properties of a gill (Na+, K+)-ATPase from the pelagic marine seabob Xiphopenaeus kroyeri. Sucrose density gradient centrifugation revealed membrane fractions distributed mainly into a heavy fraction showing considerable (Na+, K+)-ATPase activity, but also containing mitochondrial FoF1- and Na+- and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na+, K+)-ATPase a-subunit with an Mr of ≈110 kDa. The alpha-subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na+, K+)-ATPase hydrolyzed ATP obeying Michaelis-Menten kinetics with Vm = 109.5 ± 3.2 nmol Pi min-1 mg-1 and Km = 0.03 ± 0.003 mmol L-1. Mg2+ (Vm = 109.8 ± 2.1 nmol Pi min-1 mg-1, K0.5 = 0.60 ± 0.03 mmol L-1), Na+ (Vm = 117.6 ± 3.5 nmol Pi min-1 mg-1, K0.5 = 5.36 ± 0.14 mmol L-1), K+ (Vm = 112.9 ± 1.4 nmol Pi min-1 mg-1, K0.5 = 1.32 ± 0.08 mmol L-1), and NH4+ (Vm = 200.8 ± 7.1 nmol Pi min-1 mg-1, K0.5 = 2.70 ± 0.04 mmol L-1) stimulated (Na+, K+)-ATPase activity following site-site interactions. K+ plus NH4+ does not synergistically stimulate (Na+, K+)-ATPase activity, although each ion modulates affinity of the other. The enzyme exhibits a single site for K+ binding that can be occupied by NH4 +, stimulating the enzyme. Ouabain (KI = 84.0 ± 2.1 µmol L-1) and orthovanadate (KI = 0.157 ± 0.001 µmol L-1) inhibited total ATPase activity by ≈50 and ≈44 %, respectively. Ouabain inhibition increases ≈80 % in the presence of NH4 + with a threefold lower KI, suggesting that NH4+ is likely transported as a K+ congener.