Cholinesterases use a Glu - His - Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. We have previously shown by proton NMR that horse serum butyryl cholinesterase, like serine proteases, forms a short, strong hydrogen bond (SSHB) between the Glu - His pair upon binding mechanism-based inhibitors, which form tetrahedral adducts, analogous to the tetrahedral intermediates in catalysis [Viragh, C., et al. (2000) Biochemistry 39, 16200-16205]. We now extend these studies to human acetylcholinesterase, a 136 kDa homodimer. The free enzyme at pH 7.5 shows a proton resonance at 14.4 ppm assigned to an imidazole NH of the active-site histidine, but no deshielded proton resonances between 15 and 21 ppm. Addition of a 3-fold excess of the mechanism-based inhibitor m-(N,N,N-trimethylammonio)-trifluoroacetophenone (TMTFA) induced the complete loss of the 14.4 ppm signal and the appearance of a broad, deshielded resonance of equal intensity with a chemical shift δ of 17.8 ppm and a D/H fractionation factor ø of 0.76 ± 0.10, consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of δ with hydrogen bond lengths in small crystalline compounds, the length of this SSHB is 2.62 ± 0.02 Å, in agreement with the length of 2.63 ± 0.03 Å, independently obtained from ø. Upon addition of a 3-fold excess of the mechanism-based inhibitor 4-nitrophenyl diethyl phosphate (paraoxon) to the free enzyme at pH 7.5, and subsequent deethylation, two deshielded resonances of unequal intensity appeared at 16.6 and 15.5 ppm, consistent with SSHBs with lengths of 2.63 ± 0.02 and 2.65 ± 0.02 Å, respectively, suggesting conformational heterogeneity of the active-site histidine as a hydrogen bond donor to either Glu-327 of the catalytic triad or to Glu-199, also in the active site. Conformational heterogeneity was confirmed with the methylphosphonate ester anion adduct of the active-site serine, which showed two deshielded resonances of equal intensity at 16.5 and 15.8 ppm with ø values of 0.47 ± 0.10 and 0.49 ± 0.10 corresponding to average hydrogen bond lengths of 2.59 ± 0.04 and 2.61 ± 0.04 Å, respectively. Similarly, lowering the pH of the free enzyme to 5.1 to protonate the active-site histidine (pKa = 6.0 ± 0.4) resulted in the appearance of two deshielded resonances, at 17.7 and 16.4 ppm, consistent with SSHBs with lengths of 2.62 ± 0.02 and 2.63 ± 0.02 Å, respectively. The NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase from Torpedo californica complexed with TMTFA (2.66 ± 0.28 Å) and sarin (2.53 ± 0.26 Å) and at low pH (2.52 ± 0.25 Å). However, the order of magnitude greater precision of the NMR-derived distances establishes the presence of SSHBs at the active site of acetylcholinesterase, and detect conformational heterogeneity of the active-site histidine. We suggest that the high catalytic power of cholinesterases results in part from the formation of a SSHB between Glu and His of the catalytic triad.