Triplex forming oligonucleotides (TFOs) containing the nucleoside analogues 2'-O-methyl-5-propynyluridine (1) and 2'-O-methyl-5-(3-amino-1-propynyl)uridine (2) were synthesized. The affinity and selectivity of triplex formation by these TFOs were studied by gel shift analysis, T(m) value measurement, and association rate assays. The results show that the introduction of 1 and 2 into TFOs can improve the stability of the triplexes under physiological conditions. Optimized distribution of 1 or 2 in the TFOs combined with a cluster of contiguous nucleosides with 2'-aminoethoxy sugars resulted in formation of triplexes with further enhanced stability and improved selectivity.