Single-molecule force spectroscopy was applied to unfold individual Na+/H+ antiporters NhaA from membrane patches. The force–extension curves contained detailed information about the strength and location of molecular interactions established within NhaA. Although molecular interactions that stabilize secondary structure elements remained unaffected on switching NhaA into its functional state, those that are assigned to the Na+-binding site changed markedly. These interactions were formed only in the presence of Na+, with their full strength being established at pH≈6. This finding is in apparent contrast to measurements that suggest that NhaA is fully active at pH 7. Statistical analysis, however, showed that not all NhaA molecules activated this molecular interaction at pH 6, but at pH 7. This implies that the molecular interactions established on Na+ binding may represent an early step in NhaA activation. The direct observation of molecular interactions established within an antiporter provides new insights into their activation mechanisms.