The ability of oligodeoxynucleotides to form specific triple helical structures with critical regulatory sequences in the human dihydrofolate reductase (DHFR) promoter was investigated. A battery of purine-rich oligonucleotides targeted to the two purine.pyrimidine strand biased regions near the DHFR transcription initiation site was developed. The stable triple helical structures formed by binding of the oligonucleotides to the native promoter double helix were dominated by G*G.C triplets, with interspersed C*C.G and A*A.T alignments. Mismatches between the oligonucleotide and the purine-rich strand of the target significantly destabilized third strand binding, and a G*A.T alignment was particularly unfavorable. Formation of a pur.pur.pyr triple helical structure results in a localized limitation of access to the native double helical DNA and produces sequence dependent conformational alterations extending several nucleotides beyond the triplex-duplex boundary. Although they differ only by the insertion of two A.T base pairs, the distal and proximal purine.pyrimidine regions can be targeted individually due to the high degree of sequence specificity of triple helical alignment. Triplex formation overlapping any of three consensus transcriptional regulatory elements and collectively covering 50% of the DHFR core promoter is now possible with this set of oligonucleotides.