β-Cyclodextrin (β-CD) is a seven-unit cyclic sugar molecule with a torroidal shape, a hydrophilic exterior, and a hydrophobic cavity. It has been hypothesized that pyrene (P) forms a P(β-CD)2 complex in which the pyrene lies in between the two wide, or secondary, rims of the two β-CDs. This dimer structure appears to be stabilized in the crystal state by hydrogen bonds between –OH groups on the rims of the adjacent β-CDs. We have chosen to test this hypothesis by comparing the fluorescence properties of pyrene in the presence of substituted β-CDs with these properties in the presence of β-CD. These properties include the shape (I/III vibronic band ratio) and lifetime of pyrene fluorescence and the effect on the pyrene I/III ratio of additives such as an iodide ion and a fluorinated alcohol. The CD derivatives in this investigation include those with no wide rim substitution and those with substitution on both the wide and narrow rims of β-CD. Our data indicate that the a P(β-CD)2 complex forms only when there is no substitution on the wide rim, which confirms the proposed structure for this complex.