We have previously investigated the effects of removing bound Ca^(2+) ions and of adding KCl or CaCl₂ on the histidine C-2H resonances in subtilisin Carlsberg from Bacillus licheniformis by one-dimensional proton nuclear magnetic resonance spectroscopy [Korean Biochem. J. 22 (1989), 96-104]. As an extension of our previous work, effects of adding other monovalent (Li^+, Na^+, Cs^+) and divalent (Mg^(2+), Ba^(2+)) metal ions have been investigated to probe the nature of the metal ion binding sites. All monovalent ions show similar effects as do all divalent metal ions. However, the effects of monovalent and divalent ions are different from each other, particularly for the C-2H resonance of histidine 238. The results suggest that the low-affinity Ca^(2+) binding site in subtilisin Carlsberg and the adjacent monovalent cation binding site are not highly specific for any member of the divalent or monovalent metal ions, respectively, but they can differentiate between divalent and monovalent ions.