A prominent secondary four-bond hydrogen/deuterium isotope effect was observed from proton NMR at the active site histidine imidazole ring of bovine pancreatic sPLA(2) in the presence of a phosphonate transition state analogue. The cross-modulation of H(epsilon2)/H48 and H(delta1)/H48 resonances was confirmed by line shape simulation that follows the McConnell equation with fractionation factors incorporated to account for the change in the signal magnitude as well as the resonance line shape at various H(2)O/D(2)O solvent mixtures. While the downfield shift of each individual proton upon deuteration on the opposite site can be attributed to the proton-relay system of the H48-D99 catalytic dyad in sPLA(2), the observation that H(delta1)/H48 induces a 3-fold larger H/D secondary isotope effect ( approximately 0.15 ppm) on H(epsilon2)/H48 than vice versa ( approximately 0.05 ppm) is interpreted as additional spectroscopic evidence for the previously proposed short-strong hydrogen bond formed between the donor N(delta1)/H48 and a nonbridging phosphonate oxygen atom of the transition state analogue. These results provide additional details for the catalytic mechanism of sPLA(2) and demonstrate that the intrinsic H/D secondary isotope effect is a useful tool to probe hydrogen bond strength.