Phospholipase A2 [EC 3.1.1.4] treatment of pig kidney Na+,K(+)-ATPase [EC 3.6.1.3] labeled with fluorescence probes at the alpha-chain reduced the extent of the fluorescence intensity change of an N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM) probe at Cys-964 to below one-third of the control level accompanying the accumulation of phosphoenzymes. However, it only induced a slight decrease in that of a fluorescence isothiocyanate (FITC) probe at Lys-501 with a large decrease in the rate of change. The addition of phosphatidylserine (PS) or phosphatidylinositol (PI) to the phospholipase-treated BIPM-FITC-labeled enzyme increased the rate of the FITC fluorescence change. Phospholipase treatment of the BIPM-enzyme greatly reduced the Na+,K(+)-ATPase activity. The addition of PS or PI to the treated enzyme induced reactivation. These data and others suggest that Cys-964 and Glu-953 (Rb+ protectable dicyclohexyl carbodiimide binding site) are located in the vicinity of the surface area of the enzyme where hydrocarbon chains of phospholipids are present, and conserved H-bonding amino acids, Thr-955 and Ser-962, are located rather near the center of a domain forming a cation binding route or cage with other hydrophobic transmembrane segments. These data may indicate that the interaction between the BIPM probe and the hydrocarbon chains of phospholipids changes in such a way as to sense the change in the binding state of various ligands accompanying the sequential appearance of reaction intermediates of the enzyme.