We report the first parallel polarization EPR signal from the Mn(III) ion formed by photooxidation of Mn(II) bound at the high affinity Mn-binding site of photosystem II (PSII). This species corresponds to the first photoactivation intermediate formed on the pathway to assembly of the water-splitting Mn cluster. The parallel mode EPR spectrum of the photooxidation product of 1.2/1 stoichiometry Mn(II)/Mn-depleted wild-type Synechocystis sp. PCC 6803 PSII particles consists of six well-resolved transitions split by a relatively small ⁵⁵Mn hyperfine coupling (44 G). This spectral signature is absent in photooxidized Mn apoPSII complexes prepared from D1-Asp170Glu and D1-Asp170His mutants, providing direct spectral evidence for a role for this specific D1-Asp170 residue in the initial photoactivation chemistry. Temperature-dependence measurements and spectral simulations performed on the Mn(III) parallel mode EPR signal of the wild-type sample give an axial zero-field splitting value of D ≈ −2.5 cm^-1 and a rhombic zero-field splitting value of |E| ≈ 0.269 cm^-1. The negative D value for this d⁴ ion is indicative of either a ⁵B_1g symmetry ground state of an octahedral Mn(III) geometry or a ⁵B₁ symmetry ground state of a five-coordinate square-pyramidal Mn(III) geometry. The parallel mode Mn(III) EPR spectrum obtained from the wild-type photooxidized Mn apoPSII complex is contrasted with that obtained from the five-coordinate Mn(III) form of native Mn superoxide dismutase, which has a trigonal-bipyramidal geometry and a ⁵A₁ symmetry ground state giving rise to a positive D value and a much larger ⁵⁵Mn hyperfine coupling of 100 G. The D1-Asp170His mutant displays a parallel mode EPR spectrum similar to that observed in a Mn(III) model complex. The D1-Asp170Glu mutant shows no parallel mode spectrum, but in perpendicular mode it shows a broad feature near g = 5 which has spectral characteristics of an S = ³/₂ Mn(IV) ion. This suggests that this mutant provides a binding site with a less positive Mn(III)/Mn(IV) reduction potential.