Steady-state, time-resolved fluorescence spectroscopy and Molecular Mechanics (MM) were used to study the inclusion complexes of Diethyl 2,6-naphthalenedicarboxylate (DEN) with alpha- and beta-cyclodextrins (CDs). The ratio, R, of intensities of two bands that are sensitive to the medium polarity and the average of lifetime, , which is more sensitive to the medium microviscosity surrounding the guest molecule, were obtained as a function of the CD concentration and temperature. Stoichiometries, formation constants, and the changes of enthalpy and entropy upon inclusion were also obtained. The complexes prefer 1:2 (DEN:CD) stoichiometries. MM calculations were employed to study the formation of different complexes of DEN with both alpha- and betaCDs. For the most stable structure of 1: 1 complexes, an important portion of DEN is outside CD cavity, making it possible to stabilize them by adding another CD. Driving forces for 1:2 inclusion processes are dominated by nonbonded van der Waals DEN-CD interactions. Nevertheless, due to the different geometry of the 1:2 complexes, an important electrostatic interaction appears between both betaCDs in the DEN: CD, complex that does not exist between alphaCDs in the DEN:alphaCD(2) complex. Most of this contribution is due to the intermolecular hydrogen bonding formation between secondary hydroxyl groups of both betaCDs.