The use of Hoechst 33258 (HO) as a fluorescent probe to characterize the interactions between DNA and pH-sensitive amphiphiles is discussed. In the case of amine oxide amphiphiles dodecyldimethylamine oxide (DDAO) and p-dodecyloxybenzyldimethylamine oxide (pDoAO), the decrease in fluorescence emission, which signals DNA-amphiphile association, is accompanied by a large hypsochromic shift in the emission maximum of the bound probe; this eventually reaches a value characteristic of HO in a neutral or slightly basic environment. These findings are compared to results obtained by using the more common ethidium bromide (EB) probe, which shows no such shift. Circular dichroism and fluorescence depolarization experiments indicate that fluorescence emission only occurs from the DNA-bound probe, and the observed shift in emission maximum when using HO as a fluorescent probe is due to a variation in the local pH in the vicinity of the probe.