The nitrone 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been the most widely used spin trap for the detection of transient free radicals in chemical, biological, and biomedical research using electron paramagnetic resonance (EPR) spectroscopy. A density functional theory (DFT) approach was used to predict the thermodynamics of formation of the superoxide anion/hydroperoxyl radical (O-2(center dot-)/(O2H)-O-center dot) adduct of DMPO as well as its pK(a) in aqueous systems. At the B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) level, we predicted (in the gas phase and with a polarizable continuum model (PCM) for water) three conformational minima for both the DMPO-O-2(-) and DMPO-O2H adducts. Using DFT and the PCM solvation method, the pK(a) of DMPO-O2H was predicted to be 14.9 +/- 0.5. On the basis of free energy considerations, the formation of DMPO-O2H at neutral pH proceeds via initial addition of O-2(center dot-) to DMPO to form the DMPO-O-2(-) adduct and then subsequent protonation by water (or other acidic sources) to form DMPO-O2H. Under acidic conditions, the addition of (O2H)-O-center dot to DMPO is predicted to be more exoergic than the addition of O-2(center dot-) and is consistent with available experimental kinetic data.