The nitrone, 5,5-dimethylpyrroline N-oxide (DMPO), is a commonly used spin trap for the detection of superoxide radical anion (O-2(center dot-)) using electron paramagnetic resonance spectroscopy. This work investigates the reactivity of DMPO to O-2(center dot-) in mildly acidic pH (5.0-7.0). Mild acidity is characteristic of acidosis and has been observed in hypoxic systems, e.g., ischemic organs and cancer cells. Although the established pK(a) for O-2(center dot-) is 4.8, the pK(a) for DMPO is unknown. The pK(a) of the conjugate acid of DMPO was determined to be 6.0 using potentiometric, spectrophotometric, H-1 and C-13 NMR, and computational methods. H-1 and C-13 NMR were employed to investigate the site of protonation. An alternative mechanism for the spin trapping of O-2(center dot-) in mildly acidic pH was proposed, which involves protonation of the oxygen to form the N-hydroxy imino cation and subsequent addition of O-2(center dot-). The exoergicity of O-2(center dot-) addition to protonated DMPO was rationalized using density functional theory (DFT) at the PCM/B3LYP/6-31+G**//B3LYP/6-31G* level of theory.