The interaction of ethidium bromide (EB), a DNA intercalator, with two intramolecular triplexes ^5'd(G₄A₄G₄-[T₄]-C₄T₄C₄-[T₄]-G₄T₄G₄), ^5'd(G₄T₄G₄-[T₄]-G₄A₄G₄-[T₄]-C₄T₄C₄) ([T₄] represents a stretch of 4 thymine residues) and their precursor duplexes has been investigated by circular dichroism, fluorescence and UV absorption spectroscopy. Binding of EB induces a circular dichroism band in the region around 310 nm which is positive for the duplex forms but negative for the triplex forms. We observed that the binding of EB to the duplex form does not induce the formation of the triplex structures. Thermal denaturation experiments demonstrate that EB stabilizes more the parallel triple helix than the antiparallel one. Analysis of the binding process from fluorescence measurements shows that binding constants to the triple helical forms and to the hairpin reference duplex [T₄]-G₄A₄G₄-[T₄]-C₄T₄C₄) are close. However the binding site size is larger for the triplexes (4-6 base triplets) than for the duplex (2 base pairs).