The synthesis and characterization of intramolecular triple-helical DNA structures containing polyaromatic pyrene and perylene (perylenetetracarboxylic acid diimide, PDI) building blocks are presented. Two 1,8-dialkynylpyrene units are located in the WatsonCrick stem of the construct, while a PDI or a natural thymidine is present in the Hoogsteen strand. The triple helical structures were investigated by UV/VIS absorbance, fluorescence spectroscopy, and circular dichroism (CD) measurements. The folding of the intramolecular triple helix can be monitored by changes in the vibronic transition ratios, as well as by a change in the alkynylpyrene fluorescence (monomer vs. excimer). It is shown that thymine in the third strand has a pronounced influence on the interaction and, thus, on the fluorescence properties of two pyrene building blocks.