The nucleotide-binding domain (N-domain) of the Na, K-ATPase (NKA) is physicochemically characterized by a high content of Glu and Asp residues, resulting in a low isoelectric point (pI = 5.0). Acidic proteins are known to interact with cations. The analysis in silico revealed potential cation interaction sites in the NKA N-domain structure. The interaction with cations was tested in vitro by using a recombinant NKA N-domain. The N-domain contains two Trp residues at the protein surface, as determined by acrylamide-mediated fluorescence quenching, that are useful for structural studies through fluorescence changes. Intrinsic fluorescence of the N-domain was decreased by the presence of cations (Na, K, Ca) indicating an effect on the protein structure. ATP binding also decreased the N-domain intrinsic fluorescence, which allowed nucleotide affinity determination. In the presence of cations, the N-domain affinity for ATP was increased. Molecular docking of fluorescein isothiocyanate (FITC) with the N-domain showed two binding modes with the isothiocyanate group located 5-6 Å close to Lys485 and Lys506 in the nucleotide-binding site. The presence of ATP prevented the FITC covalent labeling of the N-domain demonstrating the competitive behavior for the binding site. It is proposed that cations interact with the N-domain structure and thereby modulate nucleotide (ATP) affinity and possibly affecting NKA catalysis.