Protonation of an aminoglycoside antibiotic kanamycin A sulfate was studied by potentiometric titrations at variable ionic strength, sulfate concentration and temperature. From these results the association constants of differently protonated forms of kanamycin A with sulfate and enthalpy changes for protonation of each amino group were determined. The protonation of all amino groups of kanamycin A is exothermic, but the protonation enthalpy does not correlate with basicity as in a case of simple polyamines. The sites of stepwise protonation of kanamycin A have been assigned by analysis of 1H-13C-HSQC spectra at variable pH in D2O. Plots of chemical shifts for each H and C atom of kanamycin A vs. pH were fitted to the theoretical equation relating them to pKa values of ionogenic groups and it was observed that changes in chemical shifts of all atoms in ring C were controlled by ionization of a single amino group with pKa 7.98, in ring B by ionization of two amino groups with pKa 6.61 and 8.54, but in ring A all atoms felt ionization of one group with pKa 9.19 and some atoms felt ionization of a second group with pKa 6.51, which therefore should belong to amino group at C3 in ring B positioned closer to the ring A while higher pKa 8.54 can be assigned to the group at C1. This resolves the previously existed uncertainty in assignment of protonation sites in rings B and C. © 2010 Elsevier Inc.