The assignment of the two substrate water sites of the tetra-manganese penta-oxygen calcium (Mn ₄ O ₅ Ca) cluster of photosystem II is essential for the elucidation of the mechanism of biological O-O bond formation and the subsequent design of bio-inspired water-splitting catalysts. We recently demonstrated using pulsed EPR spectroscopy that one of the five oxygen bridges (μ-oxo) exchanges unusually rapidly with bulk water and is thus a likely candidate for one of the substrates. Ammonia, a water analog, was previously shown to bind to the Mn ₄ O ₅ Ca cluster, potentially displacing a water/substrate ligand [Britt RD, et al. (1989) J Am Chem Soc 111(10):3522–3532]. Here we show by a combination of EPR and time-resolved membrane inlet mass spectrometry that the binding of ammonia perturbs the exchangeable μ-oxo bridge without drastically altering the binding/exchange kinetics of the two substrates. In combination with broken-symmetry density functional theory, our results show that ( i ) the exchangable μ-oxo bridge is O5 {using the labeling of the current crystal structure [Umena Y, et al. (2011) Nature 473(7345):55–60]}; ( ii ) ammonia displaces a water ligand to the outer manganese (Mn _A4 -W1); and ( iii ) as W1 is trans to O5, ammonia binding elongates the Mn _A4 -O5 bond, leading to the perturbation of the μ-oxo bridge resonance and to a small change in the water exchange rates. These experimental results support O-O bond formation between O5 and possibly an oxyl radical as proposed by Siegbahn and exclude W1 as the second substrate water.