The hydration of the lipid 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and of the cationic detergent dodecyltrimethylammonium bromide (DTAB) has been studied by means of isothermal titration calorimetry (ITC), gravimetry, and infrared (IR) spectroscopy. During the experiments films of the amphiphiles are perfused by an inert gas of variable relative humidity. The measurement of adsorption heats using ITC represents a new adaptation of adsorption calorimetry which has been called the humidity titration technique. This method yields the partial molar enthalpy of water upon adsorption. It is found to be endothermic with respect to the molar enthalpy of water on condensation for the water molecules which interact directly with the headgroups of POPC and DTAB. Consequently, the spontaneous hydration of the amphiphiles is entropy driven in an aqueous environment. IR spectroscopy shows that hydration is accompanied by the increase in the conformational and/or motional freedom of the amphiphilic molecules upon water binding, In particular, a lyotropic chain melting transition is induced at a certain characteristic relative humidity. This event is paralleled by the adsorption of water, The corresponding exothermic adsorption heat is consumed completely (POPC) or partially (DTAB) by the hydrocarbon chains upon melting. Differential scanning calorimetry was used as an independent method to determine transition enthalpies of the amphiphiles at a definite hydration degree. Water binding onto the headgroups is discussed in terms of hydrogen bonding and polar interactions. The adsorption isotherms yield a number of similar to 2.6 tightly bound water molecules per POPC and DTAB molecule.