Clerocidin is a complex natural molecule which induces DNA damage both directly and through irreversible/reversible poisoning of prokaryotic/eukaryotic topoisomerases II. By analysis of clerocidin reactivity towards adenine and thymine bases, we were able to fully characterize and compare the unique direct reactivity of clerocidin towards the four DNA bases, both in solution and in the DNA context. We showed that thymine was not reactive, while adenine gave a single stable covalent adduct, which was unambiguously identified as the 1,6-dialkylated species by means of modified clerocidin derivatives, modified adenine nucleotides, ESI-MS and multinuclear NMR spectroscopy. The mechanism of formation of the clerocidin adenosine adduct was similar to that occurring with cytosine, while being substantially different from that with guanine. An electrophoresis-based assay was able to highlight the unique ability of clerocidin to chemically discriminate among DNA nucleotides within a nucleic acid sequence. Finally, molecular modelling analysis gave useful indications to solve the apparent contradiction between direct and topoisomerase II-mediated covalent clerocidin reactivity with deoxyadenosine.