Calothrixin A and B are novel pentacyclic metabolites from cyanobacteria that exert growth-inhibitory effects at nanomolar concentrations against rapidly proliferating cell cultures. The exact mechanism of the biological activity has yet to be elucidated. With this in mind, we have investigated the association of the calothrixins and their synthetic analogues with various structural forms of DNA by NMR, circular dichroism (CD) and fluorescence. Calothrixin binds to DNA quadruplexes as shown by UV, CD and NMR spectroscopy but the low solubility of calothrixin in aqueous solution has been an obstacle to determining accurate dissociation constants. We have overcome this problem by incorporating a fluorescent analogue of adenine into the quadruplex formed within the hypersensitive site of the c-myc oncogene promotor. The analogue does not change the quadruplex structure but its fluorescence is very sensitive to local changes induced by the binding of other molecules. By following the changes in the fluorescence of this DNA quadruplex on binding of calothrixin, we have confirmed that calothrixin binds with micromolar dissociation constants. Further work with several different DNA quadruplex structures has confirmed that calothrixin will bind only if there is an exposed external quartet. One possible binding mode is shown below with calothrixin superposed on the exposed quadruplex quartet.