The c-myc gene is overexpressed in a variety of tumor types and appears to play an important role in the abnormal growth of a number of cell types. In an effort to determine the ability of sequence- and species-specific triplex-forming oligonucleotides to inhibit expression of a targeted gene in animals, we have identified two novel triplex-forming sites in the murine c-myc promoter. One is homologous to the triplex-forming human PuF binding element located upstream of the P1 transcription start site. The other triplex-forming site is found in a region between P1 and P2 that encompasses the ME1a1 binding site and part of the E2F binding site and is highly homologous to the human sequence. Synthetic oligodeoxyribonucleotides designed to target these essential regulatory elements form sequence-specific triple helices as demonstrated by gel mobility shift analysis and DNase I footprinting. Polypurine: polypyrimidine regions in the P1 and P2 promoters form specific protein-DNA complexes upon incubation with a murine YC8 nuclear extract. Preincubation of each of the promoter fragments with its respective triplex-forming oligonucleotide results in the inhibition of nuclear protein binding. Non-triplex-forming oligonucleotides do not significantly affect protein binding. The data presented are a preliminary step toward generating an animal model for the phenotypic effects of triplex formation within the c-myc promoter.