The inclusion complexation of phenobarbital (PHB) with 2-hydroxypropyl-B-cyclodextrin (HPCyD) in aqueous solution has been investigated by microcalorimetry, C-13-NMR spectroscopy and molecular dynamics simulations. Two different types of PHB-HPCyD inclusion complexes at 1 : 1 stoichiometry were realized by un-ionized PHB. In the first type of inclusion with higher affinity with HPCyD, the phenyl ring of PHB was included within the HPCyD cavity, whereas in the second type, the barbituric acid ring seemed to penetrate into the cavity. The ethyl side-chain remained outside of the cavity in both types. Complexation was independent of the concentrations of both PHB and HPCyD. The first type of inclusion was characterized by an entropy-driven reaction associated with constant values of Delta G(1) (-2.69+/-1.0 kJ mol(-1)), Delta H-1 (-3.73+/-0.86 kJ mol(-1)), and Delta S-1 (77.5+/-1.5 J mol(-1) K-1) at various pH, and the hydrophobic interaction dominated the stabilization of the complex. The second type was characterized by large negative values of Delta H-2 (-19.2+/-0.7 kJ mol(-1)) and small Delta S-2 (8.6+/-2.5 J mol(-1) K-1) at pH below 7.0, reflecting van der Waals' and/or electrostatic interactions, and all the thermodynamic parameters markedly decreased at pH>8.0. C-13-NMR chemical shifts of barbituric acid ring and of a phenyl ring substituted at C5 on barbituric acid ring were significantly moved upheld upon penetrating into HPCyD cavity.