We have used DNase I footprinting to assess the formation of triple helices at 15mer oligopurine target sites which are interrupted by several (up to four) adjacent central pyrimidine residues. Third strand oligonucleotides were designed to generate complexes containing central (X·TA)(n) or (X·CG)(n) triplets (X = each base in turn) surrounded by C+·GC and T·AT triplets. It has previously been shown that G·TA and T·CG are the most stable triplets for recognition of single TA and CG interruptions. We show that these triplets are the most useful for recognizing consecutive pyrimidine interruptions and find that addition of each pyrimidine residue leads to a 30-fold decrease in third strand affinity. The addition of 10 μM naphthylquinoline triplex-binding ligand stabilizes each complex so that all the oligonucleotides produce footprints at similar concentrations (0.3 μM). Targets containing two pyrimidines are only bound by oligonucleotides generating (G·TA)2 and (T·CG)2 with a further 30-fold decrease in affinity. (G·TA)2 is slightly more stable than (T·CG)2. In the presence of the triplex-binding ligand the order of stability is (G·TA)2 > (C·TA)2 > (T·TA)2 > (A·TA)2 and (T·CG)2 > (C·CG)2 > (G·CG)2 = (A·CG)2. No oligonucleotide footprints are generated at target sites containing three consecutive pyrimidines, though addition of 10 μM triplex-binding ligand produces stable complexes with oligonucleotides generating (G·TA)3, (T·CG)3 and (C·CG)3, with a further 30-fold reduction inaffinity. No footprints are generated at targets containing four Ts, though the ligand induces a weak interaction with the oligonucleotide generating (T·CG)4.