6-O-(2-Sulfonato-6-naphthyl)-γ-cyclodextrin (1) and 6-deoxy-(pyrene-1-carboxamido)-β-cyclodextrin (2) were prepared. Homodimerizations of 1 and 2 and heteroassociation between 1 and 2 were investigated by 1H NMR, circular dichroism, and fluorescence spectroscopic methods. The compounds 1 and 2 form head-to-head dimers with dimerization constants of 140 ± 50 and 270 ± 70 M -1, respectively. We also determined the association constants of 1 with β-CD as 270 ± 20 M -1 and 2 with γ-CD as 100 ± 30 M -1 from fluorescence and circular dichroism titration data, respectively. The heteroassociation between 1 and 2 was manifested in increased circular dichroism ellipticities of 2, downfield shift of the H-2 proton of the pyrene group of 2, and upfield shift of the H-5 proton of the naphthyl group of 1 upon mixing 1 and 2. The analysis of circular dichroism titration data of 2 with 1 gave the association constant as 9300 ± 1600 M -1. The NMR and circular dichroism spectra suggested that the naphthyl group of 1 is deeply included into the β-CD cavity of 2, while the pyrene group of 2 is partially inserted in the γ-CD cavity of 1 in the complex. The energy-minimized structure from molecular modeling of the complex supports this. We believe that the facile heteroassociation of two cyclodextrin derivatives having different sizes of cavity and pendant group could be utilized as a useful strategy for assembling functionalized CDs for various applications.