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The identity and rearrangements of substrate water molecules in photosystem II (PSII) water oxidation are of great mechanistic interest and addressed herein by comprehensive analysis of NH4+/NH3binding. Time-resolved detection of O2formation and recombination fluorescence as well as Fourier transform infrared (FTIR) difference spectroscopy on plant PSII membrane particles reveals the following. (1) Partial inhibition in NH4Cl buffer occurs with a pH-independent binding constant of ∼25 mM, which does not result from decelerated O2formation, but from complete blockage of a major PSII fraction (∼60%) after reaching the Mn(IV)4(S3) state. (2) The non-inhibited PSII fraction advances through the reaction cycle, but modified nuclear rearrangements are suggested by FTIR difference spectroscopy. (3) Partial inhibition can be explained by anticooperative (mutually exclusive) NH3binding to one inhibitory and one non-inhibitory site; these two sites may correspond to two water molecules terminally bound to the “dangling” Mn ion. (4) Unexpectedly strong modifications of the FTIR difference spectra suggest that in the non-inhibited PSII, ammonia binding obliterates the need for some of the nuclear rearrangements occurring in the S2–S3transition as well as their reversal in the O2formation transition, in line with the carousel mechanism [Askerka, M., et al. (2015) Biochemistry 54, 5783]. (5) We observe the same partial inhibition of PSII by NH4Cl also for thylakoid membranes prepared from mesophilic and thermophilic cyanobacteria, suggesting that the results described above are valid for plant and cyanobacterial PSII.