The structure of the DNA binding site of the Nuclear single-stranded Binding Factor (NssBF), located in the long terminal repeat of the Drosophila 1731 retrotransposon, was investigated by melting temperature experiments, chemical probing and fluorescence measurements using a macrocyclic bis-acridine. The most probable structure of this element, named Bc, mainly involves two hairpins in equilibrium at pH 6.0 at low concentration. The hairpins differ in their apical loop size; 4 and 8 nt. The structural flexibility of Bc probably derives from the three consecutive CATA repeats complementary to the GTAT nucleotides of the palindrome. In contrast, the Bc complementary strand adopts a single hairpin. Since Bc is implicated in repression of transcription via binding of two specific factors, its structural flexibility could be associated with this process.