The 2-amino group of guanine is believed to be a critical determinant of potential DNA binding sites for echinomycin and related quinoxaline antibiotics. In order to probe its importance directly we have studied the interaction between echinomycin and DNA species in which guanine N(2) is deleted by virtue of substitution of inosine for guanosine residues. The polymerase chain reaction was used to prepare inosine-substituted DNA. Binding of echinomycin, assessed by DNAse I footprinting, was practically abolished by incorporation of inosine into one or both strands of DNA. We conclude that both the purines in the preferred CpG binding site need to bear a 2-amino group to interact with echinomycin.