In the present study, five cyclic nitrone superoxide spin adducts, i.e. DMPO-OOH, M(3)PO-OOH, EMPO-OOH, DEPMPO-OOH and DEPDMPO-OOH, were chosen as model compounds to investigate the effect of 2,5-subsitituents on their stability, through structural analysis and decay thermodynamics using density functional theory (DFT) calculations. Analysis of the optimized geometries reveals that none of the previously proposed stabilizing factors, including intramolecular H-bonds, intramolecular non-bonding interactions, bulky steric protection nor the C(2)-N(1) bond distance can be used to clearly explain the effect of 2,5-substituents on the stability of the spin adducts. Subsequent study found that spin densities on the nitroxyl nitrogen and oxygen are well correlated with the half-lives of the spin adducts and consequently are the proper parameters to characterize the effect of 2,5-substituents on their stability. Examination of the decomposition thermodynamics further supports the effect of the substituents on the persistence of cyclic nitrone superoxide spin adducts.