The intermediate electron acceptor of the photosystem II (PSII) reaction center, which is a pheophytin-cr (Pheo) molecule, was studied by pulsed EPR. Electron spin echo envelope modulation (ESEEM) experiments were carried out in N-14- and N-15-labeled PSII reaction centers. The assignment of the ESEEM frequencies was achieved by the application of three-pulse (stimulated echo) and four-pulse 2D-HYSCORE sequences. Computer simulations of the N-14- and N-15-stimulated ESEEM spectra were performed, yielding a complete set of hyperfine and quadrupole coupling constants of the four nitrogens of the macrocycle of the pheophytin. The nitrogen hyperfine couplings are characterized by considerable anisotropy while the isotropic part is either smaller or much larger than the nuclear Larmor frequency : The present results show that the spin density distribution on the Pheo(-) in PSII is highly asymmetric. In agreement with earlier theoretical predictions for Pheo(-) in vitro, the ESEEM data show that in PSII the two unprotonated nitrogens bear considerable spin density while the protonated nitrogens bear a small fraction of the spin density.