To study the genotoxic properties of 1,N⁶-ethenodeoxyadenosine (εdA) in human cells, a novel site-specific mutagenesis approach was developed, in which a single DNA adduct was uniquely placed in either strand of a shuttle plasmid vector. The analysis of progeny plasmid derived from the modified strand shows that εdA, when incorporated into the position of the second A of 5′-CAA (codon 61 of the rasgene), is mutagenic in human cells, inducing A→T, A→G, and A→C mutations. The efficient induction of A→T transversions in experiments using modified double- and single-stranded DNA substrates supports the hypothesis that A:T→T:A transversions in human and animal tumors induced by vinyl compounds reflect misinsertion of dAMP opposite this adduct. Mutagenic events were similar when the adduct was incorporated into either the leading or the lagging strand. εdA was more mutagenic than 8-oxodeoxyguanosine, which induced targeted G→T transversions in HeLa cells. In Escherichia coli, εdA did not significantly miscode (<0.27%) even in the presence of induced SOS functions.