Hybrid constructions based on nucleosides and lipophilic components, known as nucleolipids, have become an extremely interesting class of molecules, especially for their potential biomedical applications. In this matter, it seemed important to define the nature and estimate the strength of their interaction with polynucleotides by different ways. We report in this work a systematic investigation through isothermal titration calorimetry of the thermodynamics of the association and dissociation of adenine and thymine derivatives, not previously performed. Then we use the results obtained on these simple systems as a basis for comparison with the binding of phospholipids functionalized with adenosine and thymidine to polyadenylic or polyuridylic acids applying the same experimental technique.