We have designed a Hoogsteen (HG) triple-helical three-way junction (ternary complex) constructed from three 33-mer oligonucleotides based on the same subset of sequences used for the Watson-Crick (WC) triple-helical three-way junction, characterized previously (P. L. Hüsler and H. H. Klump (1994) Arch. Biochem. Biophys., 313, 29-38). The junction differs primarily in the assembly of the branch point and the ends of the arms. The three oligonucleotides can each fold into a WC hairpin, linked by a four-member cytosine loop, each containing a homo-pyrimidine 10-mer single-strand extension. On lowering the pH (between 6 and 4), the extensions mutually associate to one of the other hairpins via Hoogsteen (HG) hydrogen bonding. Collectively, this process results in the formation of the branch point and the triple-helical arms. The HG triple-helical three-way junction is characterized by gel electrophoresis, circular dichroism, uv melting, and differential scanning calorimetry. The junction undergoes thermal unfolding in two distinct temperature regions. In the temperature range 15 to 50 degrees C loss of HG base pairing results in the dissociation of the three-way junction. Between 55 and 95 degrees C the resulting hairpins undergo further successive unfolding. The overall calorimetric unfolding enthalpy and entropy changes associated with the loss of HG base pairing are approximately equal to the sum of the enthalpy and entropy changes associated with the dissociation of the HG base pairing in the isolated arms (170.6 kcal.mol-1; 540.1 cal.mol-1.K-1). It is apparent from these results that in the proximity of the branch point the structure is not perturb or strain. This result is contrary to the results obtained for the WC triple-helical three-way and for three-way junctions constructed from canonical double-helical DNA. Complete folding of the junction requires either high Na+ (600 mM) ion concentrations or 40-60 mM Mg2+.