DNAase I footprinting has been used to investigate sequence selectivity in the binding of the antiviral fluorenone derivative tilorone to DNA. Using the 160 base pair EcoRI-AvaI tyr T restriction fragment and the 166 base pair EcoRI-BstEII ptyr 2 restriction fragment, obtained respectively from the Plasmids pKMΔ-98 and pMLB 1048, it is shown that tilorone binds to DNA with a preference for alternating purine-pyrimidine sequences. Enhancement of DNAase I cleavage occurs at homopolymeric A and T stretches and, to a lesser extent, at GC-rich clusters suggesting that the drug discriminates against these sequences. However, tilorone has only limited selectivity and can bind reasonably well to many types of DNA sequences. By comparison with the footprinting patterns produced by a variety of intercalating agents, it appears that tilorone protects from DNAase I cleavage the same sequences as the intercalating drug ethidium bromide. Using diethylpyrocarbohate and osmium tetroxide as probes for chemical reactivity we can perceive deformation in the structure of DNA induced by tilorone binding. Results from enzymic and chemical probing experiments are compared and discussed with respect to the likely intercalative mode of binding of tilorone to DNA.