Triplex forming oligonucleotides (TFO) are known to be potential agents for modifying gene function. In most instances they are utilized for repression of transcription. However hybrid molecules containing cis-acting elements in a duplex DNA in a hairpin form contiguously with the TFO can bind transcription factors in vitro. In the present manuscript we demonstrate that hairpin-TFO can be employed in vivo for targeted activation of gene expression of two genes mapping on chromosome XI of Saccharomyces cerevisiae. The cis-acting GAL4 protein-binding site contained in the hairpin-TFO is targeted in vivo to the 5' upstream sequence of STE6 and CBT1 genes that are transcribed in opposite directions and share a poly(pu/py) sequence that can form triple helical structure. The hairpin-TFO is targeted to this site and promotes the activation of both the genes. These results demonstrate four important aspects relating to activation of gene expression: (i) accessibility of duplex DNA packaged into chromatin to triplex forming sequences in vivo, (ii) the potential use of hairpin-TFO in therapeutics by activation of transcription in vivo, (iii) Sharing of transcription factors between two genes transcribed in opposite directions and (iv) specific activation of genes even when their cognate site is not covalently linked to the gene being activated.