A series of Roussin's red salt esters [Fe(2)(micro-RS)(2)(NO)(4)] (R = n-Pr (), t-Bu (), 6-methyl-2-pyridyl () and 4,6-dimethyl-2-pyrimidyl ()) were synthesized by the reaction of Fe(NO)(2)(CO)(2) with thiols or thiolates. Complexes were characterized by IR, UV-vis, (1)H-NMR, electrochemistry, and single-crystal X-ray diffraction analysis. The IR spectra of complexes display one weak and two strong NO stretching frequencies (nu(NO)) in solution, but only two strong nu(NO) in the solid. Density functional theoretical (DFT) calculations using complex as model suggest that two spatial isomers of these complexes bear a 3 kcal energy difference in solution. Frequency calculations of the two isomers provide insight on the origin of the vibrational bands and explain the IR observation of complexes in the solid state and in solution. Cyclic voltammetry shows two quasi-reversible, one-electron reductions for complexes and one quasi-reversible, one-electron reduction for complexes . The paramagnetic complexes [Fe(2)(micro-RS)(2)(NO)(4)](-) ((-)-(-)), which are prepared by the chemical reduction of neutral complexes [Fe(2)(micro-RS)(2)(NO)(4)] (), have also been investigated by EPR spectroscopy. Interestingly, the EPR spectra of complexes [Fe(2)(micro-RS)(2)(NO)(4)](-) ((-)-(-)) exhibit an isotropic signal of g = 1.998-2.004 without hyperfine splitting in the temperature range 180-298 K. The observations are consistent with the results of the calculations, which reveal that the unpaired electron is dominantly delocalized over the two sulfur and two iron atoms. The difference of the g values between the reduced form of Roussin's red ester and the typical dinitrosyl iron complexes is explained, for the first time, by the difference in unpaired electron distributions between the two types of complexes, which provides the theoretical bases for the use of g values as a spectroscopic tool to differentiate these biologically active complexes.