DNase I footprinting has been used to study the formation of parallel triplexes at oligopurine target sequences which are interrupted by pyrimidines at regular intervals. TA interruptions are targeted with third strand oligonucleotides containing guanine, generating G·TA triplets, while CG base pairs are targeted with thymine, forming T·CG triplets. We have attempted to optimize the stability of these complexes by varying the base composition and sequence arrangement of the target sites, and by replacing the third strand thymines with the positively charged analogue 5-(1- propargylamino)dU (U(P)). For the target sequence (AAAT)5AA, in which pyrimidines are positioned at every fourth residue, triplex formation with TG-containing oligonucleotides is only detected in the presence of a triplex- binding ligand, though stable triplexes were detected at the target site (AAAAAT)3AAAA. Triplex stability at targets containing pyrimidines at every fourth residue is increased by introducing guanines into the duplex repeat unit using the targets (AGAT)5AA and (ATGA)5AA. In contrast, placing C+·GC triplets on the 5'-side of G·TA, using the target (AGTA)5TT, produces complexes of lower stability. We have attempted further to increase the stability of these complexes by using the positively charged thymine base analogue U(P), and have shown that (TU(P)TG)5TT forms a more stable complex with target (AAAT)5AA than the unmodified third strand, generating a footprint in the absence of a triplex-binding ligand. Triplex formation at (AGTA)5AA is improved by using the modified oligonucleotide (TCGU(P))5TT, generating a complex in which the charged triplets C+·GC and U(P)·AT alternate with uncharged triplets. In contrast, placing U(P)·AT triplets adjacent to C+·GC, using the third strand oligonucleotide (U(P)CGT)5TT, reduces triplex formation, while the third strand with both substitutions, (U(P)CGU(P))5TT, produces a complex with intermediate stability. It appears that, although adjacent U(P)·AT triplets form stable triplexes, placing U(P)·AT adjacent to C+·GC is unfavorable. Similar results were obtained with fragments containing CG inversions within the oligopurine tract, though triplexes at (AAAAAC)3AA were only detected in the presence of a triplex- binding ligand. Placing C+·GC on the 5'-side of T·CG triplets also reduces triplex formation, while a 3'-C+·GC produces complexes with increased stability.