The role of chloride in photosystem II (PSII) is unclear. Several monovalent anions compete for the Cl(-) site(s) in PSII, and some even support activity. NO2(-) has been reported to be an activator in Cl(-)-depleted PSII membranes. In this paper, we report a detailed investigation of the chemistry of NO2(-) with PSII. NO2(-) is shown to inhibit PSII activity, and the effects on the donor side as well as the acceptor side are characterized using steady-state O2-evolution assays, electron paramagnetic resonance (EPR) spectroscopy, electron-transfer assays, and flash-induced polarographic O2 yield measurements. Enzyme kinetics analysis shows multiple sites of NO2(-) inhibition in PSII with significant inhibition of oxygen evolution at <5 mM NO2(-). By EPR spectroscopy, the yield of the S2 state remains unchanged up to 15 mM NO2(-). However, the S2-state g = 4.1 signal is favored over the g = 2 multiline signal with increasing NO2(-) concentrations. This could indicate competition of NO2(-) for the Cl(-) site at higher NO2(-) concentrations. In addition to the donor-side chemistry, there is clear evidence of an acceptor-side effect of NO2(-). The g = 1.9 Fe(II)-QA(-•) signal is replaced by a broad g = 1.6 signal in the presence of NO2(-). Additionally, a g = 1.8 Fe(II)-Q(-•) signal is present in the dark, indicating the formation of a NO2(-)-bound Fe(II)-QB(-•) species in the dark. Electron-transfer assays suggest that the inhibitory effect of NO2(-) on the activity of PSII is largely due to the donor-side chemistry of NO2(-). UV-visible spectroscopy and flash-induced polarographic O2 yield measurements indicate that NO2(-) is oxidized by the oxygen-evolving complex in the higher S states, contributing to the donor-side inhibition by NO2(-).