Organic synthesis and recombinant DNA techniques have been used to situate a single 1,N6-ethenoadenine (eAde) DNA adduct at an amber codon in the genome of an M13mp19 phage derivative. The deoxyhexanucleotide d[GCT(A)GC] was chemically synthesized by the phosphotriester method. Mild nonaqueous conditions were employed for deprotection because of the unstable nature of the eAde adduct in aqueous basic milieu. Physical studies involving fluorescence, circular dichroism, and 1H NMR indicated eAde to be very efficiently stacked in the hexamer, especially with the 5’-thymine. Melting profile and circular dichroism studies provided evidence of the loss of base-pairing capabilities attendant with formation of the etheno ring. The modified hexanucleotide was incorporated into a six-base gap formed in the genome of an M13mp19 insertion mutant; the latter was constructed by blunt-end ligation of d(GCTAGC) in the center of the unique Smal site of M13mp19. Phage of the insertion mutant, M13mp19-Nhel, produced light blue plaques on SupE strains because of the introduced amber codon. Formation of a hybrid between the single-strand DNA (plus strand) of M13mp19-Nhel with Smal-linearized M13mp19 replicative form produced a heteroduplex with a six-base gap in the minus strand. The modified hexamer [5’-32P]d-[GCT(eA)GC], after 5’-phosphorylation, was ligated into this gap by using bacteriophage T4 DNA ligase to generate a singly adducted genome with eAde at minus strand position 6274. Introduction of the radiolabel provided a useful marker for characterization of the singly adducted genome, and indeed the label appeared in the anticipated fragments when digested by several restriction endonucleases. Evidence that ligation occurred on both 5’ and 3’ sides of the oligonucleotide also was obtained. The adduct was introduced into a unique Nhel site, and it was observed that this restriction endonuclease was able to cleave the adducted genome, albeit at a lower rate compared to unmodified DNA. The M13mp19-Nhel genome containing eAde will be used as a probe for studying mutagenesis and repair of this DNA adduct in Escherichia coli.