Acrolein is produced extensively in the environment by incomplete combustion of organic materials, and it arises endogenously in humans as a metabolic by-product. Acrolein reacts with DNA at guanine residues to form the exocyclic adduct, 8-hydroxypropanodeoxyguanosine (HOPdG). Acrolein is mutagenic, and a correlation exists between HOPdG levels in Salmonella typhimurium treated with acrolein and a resultant increase in mutation frequency. Site-specifically modified oligonucleotides were used to explore the mutagenic potential of HOPdG in Escherichia coli strains that were either wild-type for repair or deficient in nucleotide excision repair or base excision repair. Oligonucleotides modified with HOPdG were inserted into double-stranded bacteriophage vectors using the gapped-duplex method or into single-stranded bacteriophage vectors and transformed into SOS-induced E. coli strains. Progeny phage were analyzed by oligonucleotide hybridization to establish the mutation frequency and the spectrum of mutations produced by HOPdG. The correct base, dCMP, was incorporated opposite HOPdG in all circumstances tested. In contrast, in vitro lesion bypass studies showed that HOPdG causes misincorporation opposite the modified base and is a block to replication. The combination of these studies showed that HOPdG is not miscoding in vivo at the level of sensitivity of these site-specific mutagenesis assays.