The association constants for the formation of 16 pyrimidine. purine pyrimidine triple helical complexes which vary at a single common position (Z.XY where Z = A, G, C-Me, T and XY = AT, GC, CG, TA) were determined by quantitative affinity cleavage titration. The sequence context in which the 16 pairings Z XY were studied is 5’-d(AAAAAGAGAXAGAGA)-3’ within a 242 base pair DNA fragment. The association constants of four pyrimidine rich oligodeoxyribonucleotides, 5’-d(TTTTT(Me)CT(Me)CTZT(Me)CT(Me)CT)-3’, ranged from 3.8 x 10(4) M(-1) (Z.XY = A.CG) to 5.6 x 10(7) M(-1) (Z.XY = T.AT) at 22 degrees C, pH 7.0; 10 mM bis-tris, 100 mM NaCl, 250 mu M spermine. For the sequences studied, T.AT is more stable than T.GC, T.CG, or T.TA by greater than or equal to 2.3 kcal mol(-1); C-Me+GC is more stable than C-Me.AT, C-Me.CG, or C-Me.TA by greater than or equal to 1.4 kcal mol(-1); G.TA is more stable than G.AT, G.CG, or G.GC by greater than or equal to 0.8 kcal mol(-1).