1,N⁶-Ethenodeoxyadenosine (εdA) is one of four exocyclic DNA adducts produced by chloroethylene oxide and chloroacetaldehyde, reactive metabolites of vinyl chloride, a human carcinogen. εdA has also been detected in DNA of the liver of humans and untreated animals, suggesting its formation from endogenous sources. The mutagenic potential of εdA was studied using a single-stranded shuttle vector system in several E. coli strains and in simian kidney cells (COS7). This vector system enables quantitative analysis of translesional synthesis past a site-specifically placed DNA adduct in both hosts owing to the lack of the complementary strand. In experiments with five strains of E. coli, a very limited number of targeted mutations (one εdA→T, one εdA→dC, and two εdA→single base deletion) were observed among 756 transformants in hosts preirradiated with UV; no targeted mutations were observed among 563 transformants in nonirradiated hosts. These results indicate that nonmutagenic base pairings of εdA:T are the almost exclusive events in E. coli. In COS7 cells, the frequency of targeted mutations was 70%, consisting of εdA→dG (63%), εdA→T (6%), and εdA→dC (1%), indicating that the insertion of dCMP opposite the adduct is predominant. When compared with the results for 3,N⁴-ethenodeoxycytidine (εdC), which was studied previously in the same system [Moriya et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11899−11903], the results of this study indicate that the intrinsic mutagenic potency of εdA is comparable to that of εdC in mammalian cells.