The influence of the 2-amino group of guanine on antibiotic-mediated cleavage has been studied using DNA in which that group has been removed from guanine, added to adenine, or both. A homologous series of 160 base pair fragments of DNA containing inosine and/or 2,6-diaminopurine residues in place of guanosine and/or adenine residues respectively were synthesized by the polymerase chain reaction and subjected to sequence-specific cleavage by the iron-bleomycin complex or calicheamicin gamma(1)(I). Although the 2-amino group is not absolutely required it constitutes a key structural element which directs sequence-specific cleavage of DNA. For bleomycin, relocating it created new cleavage sites at pyrimidine residues lying 3’ to 2,6-diaminopurine residues. For calicheamicin, the presence of a purine 2-amino group adjacent to the cutting site potentiated the cleavage reaction. Sequence recognition by bleomycin seems to rely on direct interaction with guanine whereas DNA conformation/flexibility appears more important for calicheamicin.