The variation of the C-D vibrational stretching frequency in primary and secondary alcohols containing the D-C-O-H functionality has been examined for cases in which the alcohol functions as a proton donor in an H-bond. The C-D stretching frequency is a function of the H-bond enthalpy of formation determined by Hartree-Fock calculations, decreasing by approximately 5 cm(-1) per kcal/mol. This decrease in frequency is attributed to the increase in the overlap of the O-H bonding electrons with the C-D antibonding orbital as the H-bond is strengthened. The Raman spectra of [1-D] trifluoroethanol and [1-D] trifluoroethoxide in aqueous solution serve as an example; the alcohol has two separate C-D stretches that differ by 45 cm(-1) and deprotonation results in an average 78 cm(-1) decrease in the C-D stretching frequency. A measured deuterium equilibrium isotope effect on the acid ionization constant of [1-D-2]trifluoroethanol of 1.13 is consistent with a decreased fractionation factor of the C-1 protons due to the decrease in the C-D stretching frequency. A model nucleoside complexed with the H-bonding residues at the active site of nucleoside hydrolase indicates that H-bond formation can explain the anomalous secondary isotope effects reported for the hydrolysis of [5'-H-3]inosine (Horenstein et al. Biochemistry 1991, 30, 10788-10795). The correlations of both the C-D stretching frequency and the fractionation factor with the conformation and H-bond strength with primary and secondary alcohols as donors should serve as tools for the characterization of these important interactions in biological systems.