The site of ionization (protonation and deprotonation) of hydroxamic acids (RCONHOH) has been investigated by heteronuclear (N-14, N-15, O-17) NMR relaxation and NOE experiments (R = Me, Ph) and ab initio theoretical methods (R = H, Me, Ph). Theoretical calculations indicate that nitrogen deprotonation is favored in all cases. Electric field gradient calculations have been used to estimate the change in nuclear quadrupolar coupling constants at O and N upon ionization and compared to experimental line width changes. NMR relaxation rate and NOE measurements in aqueous solution indicate that acetohydroxamic acid (R = Me) in water is predominantly an oxygen acid, whereas benzohydroxamic acid (R = Ph) is predominantly a nitrogen acid in methanol. Acetohydroxamic acid (m* = 0.25, pK(BH+) = -1.15) is protonated at the carbonyl oxygen.