Pyrimidine oligonucleotides bind to the major groove of an oligopyrimidine-oligopurine DNA sequence by triple helix formation. A 14-mer oligopyrimidine 3'-psoralen-conjugate (P) and a doubly modified 5'-acridine/3'-psoralen-oligonucleotide (PA) were photo-crosslinked to their target site. The crosslinked complexes were tested regarding their sensitivity to Uvr(A)BC excinuclease/DNA complex formation and excision, and compared to free psoralen crosslinked to the same site (M). An electrophoretic mobility-shift assay showed that the crosslinked triple-helix did not hamper formation of the (A) 2 B complex under conditions where the third strand was bound to its target. In vitro excision experiments performed on damaged DNA fragments containing crosslinked 5-methoxypsoralen (M-target) confirmed that the psoralen photoadduct was recognized by Uvr(A)BC and that excision occurred at the crosslinked site. The major cleavage reaction took place on the 5'-side of oligopurine strand. The excision was less efficient on the 5'-side of the pyrimidine strand. The 3'-side incision either on the purine or pyrimidine strand was even weaker. With optimal Uvr(A) concentrations, it was observed that the incision reaction on (P)- and (PA)-modified targets was clearly inhibited compared to the (M)-modified target, reflecting an effect of the oligonucleotide on the recognition/excision process. These results demonstrate that a triple helix is efficient in promoting inhibition of Uvr(A)BC excision nuclease activity. These results could account for divergent findings concerning the effects of triple helix-forming oligonucleotides on repair systems and open new perspectives to study DNA repair processes through the use of bi-substituted triple helix-forming oligonucleotides.