Stimuli-responsive molecular assemblies of potential drug/guest molecules through noncovalent host-guest interaction have been found very attractive in transporting and releasing the desired form on demand. In this article, the host-guest interaction of a drug, Hoechst-33258 (H33258), has been investigated in aqueous solutions in the presence of a macrocyclic host, namely, cucurbit[8]uril (CB8). The pH dependent structural conformations of H33258 are found to be decisive in determining the stoichiometry and geometry of the supramolecular assembly. Interaction of CB8 with the monocationic H33258 at pH 7.2 is very strong with an overall binding constant of the order of 10(11) M(-2). The noncovalently stabilized assembly with 2:1 (CB8:H33258) stoichiometry brings out ∼26-fold enhancement in the emission yield. On the other hand, the strong ion-dipole interactions provided by the dicationic dye at pH 4.5 support the CB8 to uptake two dicationic H33258 dyes in its cavity in a 1:2 stoichiometry (K(pH 4.5) = (3.2 ± 0.2) × 10(11) M(-2)). In this case, the fluorescence displayed a quenching with a decrease in the emission yield from 0.4 to 0.2. The distinct pH-mediated stoichiometric switching of CB8-H33258 complexes and the contrasting fluorescence properties demonstrated here would find application in the field of biomolecular imaging and exchange of included guests for a selective drug transport/release.