Efficient proton removal from the oxygen-evolving complex (OEC) of photosystem II (PSII) and activation of substrate water molecules are some of the key aspects optimized in the OEC for high turnover rates. The hydrogen-bonding network around the OEC is critical for efficient proton transfer and for tuning the position and pKa values of the substrate water/hydroxo/oxo molecules. The D1-N181 residue is part of the hydrogen-bonding network on the active face of the OEC. D1-N181 is also associated with the chloride ion in the D2-K317 site and is one of the residues closest to a putative substrate water molecule bound as a terminal ligand to Mn4. We studied the effect of the D1-N181A and D1-N181S mutations on the water oxidation chemistry at the OEC. PSII core complexes isolated from the D1-N181A and D1-N181S mutants have steady-state O2 evolution rates lower than those of wild-type PSII core complexes. Fourier transform infrared spectroscopy indicates slight perturbations of the hydrogen-bonding network in D1-N181A and D1-N181S PSII core complexes, similar to the effects of some other mutations in the same region, but to a lesser extent. Unlike in wild-type PSII core complexes, a g=4 signal was observed in the S2-state EPR spectra of D1-N181A and D1-N181S PSII core complexes in addition to the normal g=2 multiline signal. The S-state cycling of D1-N181A and D1-N181S PSII core complexes was similar to that of wild-type PSII core complexes, whereas the O2-release kinetics of D1-N181A and D1-N181S PSII core complexes were much slower than the O2-release kinetics of wild-type PSII core complexes. On the basis of these results, we conclude that proton transfer is not compromised in the D1-N181A and D1-N181S mutants but that the O-O bond formation step is retarded in these mutants.