The steady-state kinetics of CO2 hydration catalyzed by human carbonic anhydrase I (carbonate hydro-lyase, EC 4.2.1.1) has been investigated at three pH values corresponding to different parts of the pH-rate profile. Two buffer systems with similar pKa values were used at each pH. The results show that the catalyzed rates depend on the buffer concentration but also on the chemical nature of the buffer. For example, at pH 8.8 the buffer 1,2-dimethylimidazole behaves formally as a second substrate in a 'ping-pong' mechanism yielding a maximal kcat value of 2.2 x 10(5) s-1, whereas much lower rates were obtained with Taps buffers. Similarly, at pH 7.3 1-methylimidazole yields higher rates than Mops and at pH 6.3 3,5-lutidine is more efficient than Mes. Non-Michaelis-Menten kinetics were observed with all buffers except 1,2-dimethylimidazole. In addition, while the apparent buffer activation by 1,2-dimethylimidazole can be described by a single Km value of 26 mM, the Mes concentration dependence is consistent with the presence of two components of similar magnitudes with Km values of 45 mM and 0.15 mM. These results are interpreted within the framework of the 'zinc-hydroxide' mechanism in terms of multiple pathways for the rate-contributing transfer of a proton from the zinc-bound water molecule, formed during CO2/HCO3- interconversion, to the reaction medium, thus, regenerating zinc-bound OH-.