It was observed that a group of unusually stable DNA hairpins (Hn: 5´-d-(AG)_nT₄(CT)_n, n = 2-4) were directed to homopyrimidine sequences (Pn: 5´-d-(TC)_n) by py · pu · py-type triplex formation, resulting in high binding affinity and specificity. The spectroscopic results (UV and CD) showed that the short bimolecular triplex Hn:Pn could be formed in acidic conditions (pH 4.5-6.0) as helix length n > 2, and further extending to neutral pH as n = 4. This hairpin strategy for recognition of a pyrimidine strand has a substantial binding advantage over either the conventional linear analog or simple Watson-Crick complement. Triplex stability of Hn with Pn is not only pH-dependent, as expected for triplexes involving C⁺ · GC triads, but also sensitive to the buffer. The triplex H4:P4 was formed in the phosphate buffers of pH 6.0-7.0 but already dissociated above pH 6.5 in the buffer of cacodylate, MOPSO or PIPES. By contrast, the nature of a buffer had no major influence on stability of a hairpin duplex. Here we provide a simple triplex system, and the data presented here may be useful in defining the experimental conditions necessary to stabilize triplex DNA.