The ability of a histidine residue at position 67 in human carbonic anhydrase III to transfer protons in the catalytic pathway for the hydration of CO2 was investigated for a series of site-specific mutants. Wild-type carbonic anhydrase III has an arginine at this position with the C alpha of residue 67 about 9.4 A from the zinc. The active-site cavity contains no other residues capable of facile proton transfer. Rate constants for proton transfer from His 67 to the zinc-bound hydroxide were determined from the rate constants for the exchange of 18O between CO2 and water measured by mass spectrometry. A range of values for the pKa of zinc-bound water was achieved by replacement of phenylalanine with leucine and aspartate at position 198 adjacent to the zinc. Application of Marcus rate theory showed that intramolecular proton transfer involving His 67 had an intrinsic energy barrier of 1.3 +/- 0.3 kcal/mol and a thermodynamic work function for a preceding unfavorable equilibrium of 10.9 +/- 0.1 kcal/mol. We previously showed that proton transfer from histidine 64 in carbonic anhydrase III could be described by Marcus rate theory [Silverman, D. N., Tu, C. K., Chen, X., Tanhauser, S. M., Kresge, A. J., & Laipis, P. J. (1993) Biochemistry 32, 10757-10762]. In comparison, proton transfer from His 67 must overcome a more unfavorable preceding equilibrium (a larger work function) that probably represents an energy requirement for proper alignment of donor and acceptor groups plus the intervening hydrogen-bonded water. Once this alignment is achieved, the intrinsic energy barrier appears the same for His 67 or His 64.