Solvent effects on magnetic parameters of nitroxide spin labels in combination with side-directed spin-labeling EPR methods provide very useful means for elucidating polarity profiles in lipid bilayers and mapping local electrostatic effects in complex biomolecular systems. One major contributor to these solvent effects is the hydrogen bonds that could be formed between the nitroxide moiety and water and/or the available hydroxyl groups. Here, formation of hydrogen bonds between a lipid bilayer spin probe5-doxyl stearic acid, 5DSAand hydrogen-bond donors has been studied using high-frequency (HF) pulsed ENDOR and EPR. A hydrogen-bonded deuteron was directly detected in HF ENDOR (130 GHz) spectra of 5DSA dissolved in several deuterated alcohols, while the characteristic signal was absent in nonpolar toluene-d(8). The length of the hydrogen bond, 1.74 +/- 0.06 A, and its geometry were found to be essentially the same for all four alcohols studied, indicating that nearly identical hydrogen bonds have been formed regardless of the solvent dielectric constant. This strengthens a hypothesis that HF EPR spectra are exclusively sensitive to formation of hydrogen bonds and could be used for probing the hydrogen-bond network in complex biomolecular assemblies and lipid bilayers with site-directed spin-labeling methods.