We have prepared DNA fragments containing the sequences A15CGT15,T15and T(AT)8CG(AT)15 cloned within the Smal site of the pUC19 poly I Inker. These have been used as substrates in footprinting experiments with DNase I and diethylpyrocarbonate probing the effects of echinomycin, binding to the central CG, on the structure of the surrounding sequences. No clear DNase I footprints are seen with T15CGA15 though alterations in the nuclease susceptibility of surrounding regions suggest that the ligand is binding, albeit weakly at this site. All the other fragments show the expected footprints around the CG site. Regions of An and Tn are rendered much more reactive to DNase I and adenines on the 3′-side of the CG become hyperreactive to diethylpyrocarbonate. Regions of alternating AT show unusual changes in the presence of the ligand. At low concentrations (5μM) cleavage of TpA is enhanced, whereas at higher concentrations a cleavage pattern with a four base pair repeat is evident. A similar pattern is seen with micrococcal nuclease. Modification by diethylpyrocarbonate is strongest at alternate adenines which are staggered in the 5′-direction across the two strands. We interpret these changes by suggesting secondary drug binding within regions of alternating AT, possibly to the dinucleotide ApT. DNase I footprinting experiments performed at 4°C revealed neither enhancements nor footprints for flanking regions of homopolymeric A and T suggesting that the conformational changes are a necessary consequence of drug binding.