The acidity constants of H2-(AMPS)± were determined by potentiometric pH titrations in aqueous solution at 25°C and I=0.1M (NaNO3). Titrations with a combined single-junction glass electrode were hampered in the presence of AMPS by a “poisoning” effect; the problem could be avoided by use of two separated electrodes. The values of the acidity constants PKHH2(AMPS) = 3.72 ± 0.03 and pKHH2(AMPS) = 4.83 ± 0.02 are relatively close to each other; the buffer regions of the two equilibria overlap, and therefore a micro acidity constant scheme was developed and the constants for the various sites calculated. It is concluded that the thiophosphateprotonated species (AMPS⋅H)− dominates at about 75% occurrence, while the form (H⋅AMPS)−, with the proton at the N1 site of the adenine residue, occurs at about 25%. Semiempirical AM1 and PM3 calculations including water as a solvent locate the proton in (AMPS⋅H)− mainly on the terminal oxygen atoms rather than the sulfur. The acid-base properties of H2(AMPS)± are considerably more complicated than those of the parent nucleotide, H2(AMP)±; for the latter the two (intrinsic) acidity constants are well separated and consequently practically all protons have left the N1 site before deprotonation at the monoprotonated phosphate group occurs. Finally, an estimate for the acidity constants of H2(ATPγS)2- is given.