Triplex-forming oligonucleotides (TFOs) recognize and bind to specific duplex DNA sequences and have been used extensively to modify gene function in cells. Although germ line mutations can be incorporated by means of embryonic stem cell technology, little progress has been made toward introducing mutations in somatic cells of living organisms. Here we demonstrate that TFOs can induce mutations at specific genomic sites in somatic cells of adult mice. Mutation detection was facilitated by the use of transgenic mice bearing chromosomal copies of the supF and cII reporter genes. Mice treated with a supF-targeted TFO displayed about fivefold greater mutation frequencies in the supF gene compared with mice treated with a scrambled sequence control oligomer. No mutagenesis was detected in the control gene (cII) with either oligonucleotide. These results demonstrate that site-specific, TFO-directed genome modification can be accomplished in intact animals.