Pyrrole-imidazole polyamides are synthetic ligands that bind predetermined DNA sequences with subnanomolar affinity. We report the synthesis and characterization of an eight-ring hairpin polyamide conjugated at the turn to both enantiomers of 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI), an alkylating moiety related to CC-1065. Alkylation yields and specificity were determined on a restriction fragment containing six base pair match and mismatch sites. Alkylation was observed at a single adenine flanking the polyamide binding site, and strand selective cleavage could be achieved based on the enantiomer of seco-CBI chosen. At 1 nM concentrations of polyamide-seco-CBI conjugate, near quantitative cleavage was observed after 12 h. These bifunctional molecules could be useful for targeting coding regions of genes and inhibition of transcription.