The hydrogen-bond (H-bond) donating strengths of a series of 36 hydroxylic H-bond donors (HBDs) with N-methylpyrrolidinone have been measured in CCl4 solution by FTIR spectrometry. These data allow the definition of a H-bond acidity scale named pK(AHY) covering almost three pK units, corresponding to 16 kJ mol(-1). These results are supplemented by equilibrium constants determined in CH2Cl2 for one-third of the data set to study compounds showing a poor solubility in CCl4. A systematic comparison of these experimental results with theoretical data computed in the gas phase using DFT (density functional theory) calculations has also been carried out. Quantum electrostatic parameters appear to accurately describe the H-bond acidity of the hydroxyl group, whereas partial atomic charges according to the Merz-Singh-Kollman and CHelpG schemes are not suitable for this purpose. A substantial decrease of the H-bond acidity of the OH group is pointed out when the hydroxyl moiety is involved in intramolecular H-bond interactions. In such situations, the interactions are further characterized through AIM and NBO analyses, which respectively allow localizing the corresponding bond critical point and the quantification of a significant charge transfer from the available lone pair to the sigma(OH)* antibonding orbital. Eventually, the H-bond ability of the hydroxyl groups of steroid derivatives and of lateral chains of amino acids are evaluated on the basis of experimental and/or theoretical data.