Synthetic triple helix-forming oligodeoxyribonucleotides (TFOs) have been used to alter gene expression and to induce targeted genome modification in cells and animals. However, the efficacy of such oligodeoxyribonucleotides (ODNs) depends on efficient intracellular delivery. A novel vector system was tested for the production of single-stranded DNA (ssDNA) to serve as a TFO in mouse cells. Mouse cells carrying a substrate that can report triplex-stimulated intrachromosomal recombination were transfected with a series of ssDNA vectors, and induced recombination was assayed. Transfection with a vector set designed to generate a 34 nt G-rich ssDNA capable of triplex formation at a 30 bp polypurine target site within the reporter substrate yielded recombinants at a frequency of 196 x 10(-6), versus a background frequency of 45 x 10(-6) in mock transfected cells. No induction was seen when a vector set lacking the TFO sequence insert was tested or when the component vectors were transfected individually. Vectors engineered to express a C-rich 34 nt sequence (not expected to form triplex under physiological conditions) had no effect over background. Primer extension analyses on lysates from transfected cells confirmed the production of the intended ssDNAs. These results suggest that ssDNA molecules of a defined sequence can be generated intracellularly using a novel vector system and that such molecules are active in mediating triplex-dependent chromosomal events. The ability to produce active TFOs within cells may provide a new foundation for triplex-based gene targeting strategies.