Dimethyldiazaperopyrenium is one of the largest known DNA intercalators. Fluorescence energy transfer occurred between dimethyldiazaperopyrenium (donor) and ethidium (acceptor) when these dyes were bound to a double-stranded polynucleotide such as poly d(A-T). The addition of increasing amounts of ethidium bromide led to a marked shortening of the fluorescence lifetime of the donor, whereas the excited state of the acceptor was progressively populated via energy transfer from the donor. Critical Förster distance between these two chromophores was calculated to be 3.8 nm. The observed transfer efficiency was lower than that calculated on the basis of this critical distance and a statistical distribution of bound drugs. These results are discussed taking into account the conformational change induced by intercalation of dimethyldiazaperopyrenium in the double-stranded polynucleotide.