A combined electron microscopic and graded tryptic dissection analysis of the catalytic protein of pure Na,K-motive ATPase reveals that the aspartyl phosphate residue-carrying catalytic center is located at the utmost radius of the hydrophilic cytoplasmic domain. This makes the cation-phosphate symport mechanism of Na/K transport across plasma membrane as proposed by Mitchell topologically unrealistic, and indirectly favors the mechanism of conformational coupling between the catalytic and ionophoric function of the transport system.