Controller proteins such as C.AhdI regulate the expression of bacterial restriction–modification genes, and ensure that methylation of the host DNA precedes restriction by delaying transcription of the endonuclease. The operator DNA sequence to which C.AhdI binds consists of two adjacent binding sites, OL and OR. Binding of C.AhdI to OL and to OL + OR has been investigated by circular permutation DNA-bending assays and by circular dichroism (CD) spectroscopy. CD indicates considerable distortion to the DNA when bound by C.AhdI. Binding to one or two sites to form dimeric and tetrameric complexes increases the CD signal at 278 nm by 40 and 80% respectively, showing identical local distortion at both sites. In contrast, DNA-bending assays gave similar bend angles for both dimeric and tetrameric complexes (47 and 38°, respectively). The relative orientation of C.AhdI dimers in the tetrameric complex and the structural role of the conserved Py-A-T sequences found at the centre of C-protein-binding sites are discussed.