The ¹⁸O exchange rates for the substrate water in photosystem II can be obtained by time-resolved mass spectrometric approaches and provide insight into the chemical nature of the substrate binding sites in the oxygen evolving complex (OEC). In this communication, we report additional data on the resolvable S-state dependence of the substrate exchange rates in untreated spinach thylakoids. The data reveals that the exchange for one substrate water molecule is resolvable in all S states, i.e. S₀ (k₁ ≈ 10 s⁻¹), S₁ (k₁ ≈ 0.02 s⁻¹), S₂ (k₁ ≈ 2.0 s⁻¹) and S₃ (k₁ ≈ 2.0 s⁻¹), and that the second substrate water molecule is resolvable in S₂ (k₂ ≈ 120 s⁻¹) and S₃ (k₂ ≈ 40 s⁻¹) states, and faster than 120 s⁻¹ during the S₀ and S₁ states. The temperature dependencies of the exchange rates are also reported. The corresponding activation energies for the slow exchange substrate water in S₁, S₂ and S₃ are 83 ± 4, 71 ± 9, and 78 ± 9 kJ mol⁻¹, respectively. In contrast, the activation energy for the fast exchange substrate water in S₃, is 40 ± 5 kJ mol⁻¹. Finally we also report the secondary isotope effect of deuterium on S₃ state measurements, where the slow exchange substrate water remains unaffected while the rate for the fast exchange substrate water increases by a factor of ∼35%, resulting in an inverse H/D isotope effect. The exchange rates, activation energies and deuterium effects provide further mechanistic constraints on the water oxidation reaction in photosystem II and provide strong evidence that the slow exchange substrate water does not undergo any significant changes in its binding properties during the S₂–S₃ transition.