Monolayers of Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) were prepared and investigated on distilled water, various substrates (Na+, K+, ATP and an ATP-MgCl2 mixture) and inhibitor (ouabain). The monolayer area of the Na+,K+-ATPase is increased upon binding of Na+ and K+ (especially in the concentration range 5-20 mM of these cations in aqueous subphase). The ratio DELTAA(Na+)/DELTAA(K+) of these increases at 30 mN m-1 for the Na+,K+-ATPase on 20-100 mM subphases of NaCl and KCl is close to 3:2, which coincides with the well-known relative binding capacity of the Na+,K+-ATPase for these cations. The parameters of the Na+,K+-ATPase monolayers depend strongly on the concentration of ATP disodium salt and ATP-MgCl2 mixture in the aqueous subphases, but are almost independent of the concentration of ouabain. As was revealed by Brewster angle microscopy, the Na+,K+-ATPase monolayers on-water and ouabain have a similar domain structure at low surface pressures (0.1-0.5 mN m-1), which transforms to an isotropic homogeneous phase upon further compression. The monolayers of the Na+,K+-ATPase on NaCl, KCl, ATP and its mixture with MgCl2 show no features detectable in the microscope even at low surface pressures. All these data allow us to assume that the Na+,K+-ATPase is oriented in the monolayers predominantly with the cytoplasmic side towards the aqueous subphase and only on K+, probably, with the extracellular side towards the aqueous subphase.