Native and azide reacted Cu, Zn superoxide dismutase in aqueous and mixed water-glycerol solution have been investigated by EPR spectroscopy at low temperature. An accurate computer simulation, based on a well established theoretical model which has been reformulated for rhombic symmetry, has shown that the EPR spectrum of the copper ion in the native protein shows a significant g and A strain in the parallel region. The strain arises from a distribution of the ligand field strengths onto the metal ion and this could be traced back to the existence of a multiplicity of conformational states in the protein molecule. The strain is reduced in the presence of azide which is known to bind directly to the copper atom and to give rise to a more relaxed configuration corresponding to a square pyramidal geometry in which the apical ligand occupies an elongated position. In both samples, addition of glycerol further reduces the strain, indicating that the solvent is directly coupled to the protein matrix, thereby modulating the structural heterogeneity displayed by the protein molecule.