The zinc binding chelate system of carbonic anhydrase contains two groups of labile protons; those on the zinc bound water moIecuIe and those on the pyrrole type nitrogen of the imidazole ligands. As there is strong evidence to support the isolation of the activity generating dissociation to the chelate system, an inhibitor study has been carried out to determine which of the two groups is responsible. The open coordination position on the essential zinc ion of bovine carbonic anhydrase B has been probed through the use of uncharged inhibitor molecules. Methanol inhibits by binding in a one to one stoichiometry to the enzyme. The equilibrium dissociation constant Kj inflects from a value near 3 M at pH 5.3 to a value of 0.9 M at pH 7.6. The equivalence point, located at pH 6.4, has tbe characteristics of the pK,,z, shifting to higher pHin the presence of small concentrations of monovatent anions. As wetI, it would appear that the site of the methanol inhibition is identical to, or at least sterically overlapping with. tbe site occupied by the inhibitory monovalent anions. Aniline, lie methanol, interacts with the metal ion at a single binding site. The inhibition as given by the Ki is constant below pH 7, at 22 mhi. At higher pH values, the binding affmity falls off, governed by the deprotonation of a single group with a pKa near 7.2.