The H-1 NMR spectra of the oxidized high-potential iron-sulfur protein (HiPIP) I from Ectothiorhodospira halophila have been investigated, and the sequence-specific assignment of the protons of the cluster-bound cysteines has been performed. The spectrum of this protein is different from those of the six other proteins investigated to date in that at least one hyperfine shifted signal is missing. It is demonstrated that the "missing" signals of the beta CH2 protons of Cys 66 are under the diamagnetic envelope at 2.60 and 6.30 ppm (at 288 K). The hyperfine shifts of these protons and those of the beta CH2 protons of Cys 36 are consistent with the proposition that the oxidized cluster is in equilibrium between two electronic species. These species differ with respect to their valence distribution within the protein frame and are present in a molar ratio of about 4:1 at room temperature. An empirical relationship between the hyperfine shifts of the beta CH2 protons of Cys 36 and 66 and the percentage of the two electronic species is proposed.