We evaluate hemolymph osmotic and ionic regulatory abilities and characterize a posterior gill microsomal (Na +, K +)-ATPase from the marine swimming crab, Callinectes ornatus, acclimated to 21‰ or 33‰ salinity. C. ornatus is isosmotic after acclimation to 21‰ but is hyposmotic at 33‰ salinity; hemolymph ions do not recover initial levels on acclimation to 21‰ salinity but are anisoionic compared to ambient concentrations, revealing modest regulatory ability. NH 4 + modulates enzyme affinity for K +, which increases 187-fold in crabs acclimated to 33‰ salinity. The (Na +, K +)-ATPase redistributes into membrane fractions of different densities, suggesting that altered membrane composition results from salinity acclimation. ATP was hydrolyzed at maximum rates of 182.6 ± 7.1 nmol Pi min − 1 mg − 1 (21‰) and 76.2 ± 3.5 nmol Pi min − 1 mg − 1 (33‰), with little change in K M values (≈ 50 µmol L − 1 ). K + together with NH 4 + synergistically stimulated activity to maximum rates of ≈ 240 nmol Pi min − 1 mg − 1 . K I values for ouabain inhibition (≈ 110 µmol L − 1 ) decreased to 44.9 ± 1.0 µmol L − 1 (21‰) and 28.8 ± 1.3 µmol L − 1 (33‰) in the presence of both K + and NH 4 +. Assays employing various inhibitors suggest the presence of mitochondrial F 0F 1-, and K +- and V-ATPase activities in the gill microsomes.