We have used DNase I footprinting to examine the formation of intermolecular triplexes on DNA fragments which have been complexed with nucleosome core particles. We have prepared five DNA fragments, based on the 160-bp tyrT sequence, which contain different length oligopurine tracts (up to 25 bp) at two different positions along the fragment, and have examined their availability for triple-helix formation after reconstituting onto nucleosome core particles. These results are compared with the formation of shorter triplexes in the same regions. In general we find that increasing the length of the complex does not facilitate nucleosomal triplex formation and that the most important factor affecting triplex formation is the position of the target site within the nucleosome-bound fragment. In some instances we find that longer oligonucleotides inhibit triplex formation. Although successful triplex formation was achieved on the longest nucleosome-bound oligopurine tracts, this was accompanied by changes in cleavage pattern that suggest oligonucleotide-induced changes in nucleosome structure.