The energetics of cooperative binding by oligodeoxyribonucleotides to adjacent sites by triple helix formation have been determined as a function of sequence composition at the junction. The binding affinity of an 11-mer in the presence of a neighboring bound oligonucleotide is enhanced by factors of 12, 17, 61, and 127 when 5’-TT-3’, 5’-(CC)-C-m-C-m-3’, 5’-(TC)-C-m-3’, and 5’-(CT)-C-m-3’ stacks, respectively, are formed at the junction (10 mM Bis-Tris . HCl at pH 7.0, 10 mM NaCl, 250 mu M spermine, 24 degrees C). These binding enhancements correspond to interaction energies between the two oligonucleotides of 1.5, 1.7, 2.5, and 2.9 kcal . mol(-1), respectively. The energetic penalties for a single-base mismatch differ depending on sequence and the location of the mismatch with respect to the 5’- or 3’-side of the junction. In the case of a 5’-TT-3’ stack, a T . GC mismatch on the 5’-side of the junction decreases the interaction energy from 1.5 to 0.6 kcal . mol(-1), whereas a T . GC mismatch on the 3’-side destroys cooperativity. For a 5’-(CT)-C-m-3’ stack, a C-m . AT mismatch on the 5’-side of the junction decreases the cooperative interaction energy from 2.9 to 1.7 kcal . mol(-1), whereas a T . GC mismatch on the 3’-side of the junction destroys cooperativity. Two 11-mer oligonucleotides interacting through a 5’-TT-3’ stack binding to adjacent sites on DNA are significantly more sensitive to single-base mismatches than the corresponding 22-mer binding to the same two abutting sites.