We have studied the formation of DNA triple helices in different sequence contexts and show that, for the most stable triplexes, their apparent stability is affected by the stability of the underlying duplex. For a 14-mer parallel triplex-forming oligonucleotide (generating C(+).GC and T.AT triplets) at pH 5.0 the T(m) is more than 10 degrees C lower with an intermolecular 14-mer duplex target, than it is with an intramolecular duplex, or one which is flanked by 6 GC base pairs at either end. A similar effect is seen with triplex-forming oligonucleotides that contain stabilising analogues, for which the T(m) is higher for an intramolecular than an intermolecular duplex target. These results suggest that the use of simple intermolecular duplex targets may underestimate the triplex stabilisation that is produced by some nucleotide analogues.