The magnetic properties of the Rieske protein purified from Chlorobaculum tepidum were investigated using electron paramagnetic resonance and hyperfine sublevel correlation spectroscopy (HYSCORE). The g-values of the Fe2S2 center were g(x) = 1.81, g(y) = 1.90, and g(z) = 2.03. Four classes of nitrogen signals were obtained by HYSCORE. Nitrogens 1 and 2 had relatively strong magnetic hyperfine couplings and were assigned as the nitrogen directly ligated to Fe. Nitrogens 3 and 4 had relatively weak magnetic hyperfine couplings and were assigned as the other nitrogen of the His ligands and peptide nitrogen connected to the sulfur atom via hydrogen bonding, respectively. The anisotropy of nitrogen 3 reflects the different spin density distributions on the His ligands, which influences the electron transfer to quinone.