The oxidizing side of photosystem II contains two redox-active tyrosyl side chains, Tyrz and TyrD, and a cluster of Mn atoms involved in water oxidation. The structural environment of these components is unknown, and with computer-assisted modeling we have created a three-dimensional model for the structures around Tyrz and TyrD [Svensson et al. (1990) EMBO J. 9, 2051–2059]. Both tyrosines are proposed to form hydrogen bonds to nearby histidine residues (for Synechocystis 6803, these are His 190 on the D1 and His 189 on the D2 proteins). We have tested this proposal by electron paramagnetic resonance (EPR) spectroscopy of TyrDox in mutants of the cyanobacterium Synechocystis 6803 carrying site-directed mutations in the D2 protein. In two mutants, where His 189 of the D2 protein is changed to either Tyr or Leu, the normal EPR spectrum from TyrDox is replaced by narrow, structureless radical signals with g-values similar to that of TyrDox (g ≈ 2.0050). The new radicals copurify with photosystem II, are dark-stable, destabilized by elevated pH, and light-inducible, and originate from radicals formed by oxidation. These properties are similar to those of normal TyrDox, and we assign the new spectra to TyrDox in an altered environment induced by the point mutation in His 189. In a third mutant, where G1n 164 of the D2 protein was mutated to Leu, we also observed a modified EPR spectrum from TyrDox. This is also consistent with the model in which this residue is found in the immediate vicinity of TyrDox. Thus the results provide experimental evidence supporting essential aspects of the structural model.