We have compared hydrogen bond lengths on enzymes derived with high precision (< or = +/- 0.05 A) from both the proton chemical shifts (delta) and the fractionation factors (phi) of the proton involved with those obtained from protein X-ray crystallography. Hydrogen bond distances derived from proton chemical shifts were obtained from a correlation of 59 O--H....O hydrogen bond lengths, measured by small molecule high-resolution X-ray crystallography, with chemical shifts determined by solid-state nuclear magnetic resonance (NMR) in the same crystals (McDermott A, Ridenour CF, Encyclopedia of NMR, Sussex, U.K.: Wiley, 1996:3820-3825). Hydrogen bond distances were independently obtained from fractionation factors that yield distances between the two proton wells in quartic double minimum potential functions (Kreevoy MM, Liang TM, J Am Chem Soc, 1980;102:3315-3322). The high-precision hydrogen bond distances derived from their corresponding NMR-measured proton chemical shifts and fractionation factors agree well with each other and with those reported in protein X-ray structures within the larger errors (+/-0.2-0.8 A) in distances obtained by protein X-ray crystallography. The increased precision in measurements of hydrogen bond lengths by NMR has provided insight into the contributions of short, strong hydrogen bonds to catalysis for several enzymatic reactions.