We have previously shown that the pyrimidine oligonucleotide 5′CTTCCTCCTCT (Y11) recognizes the double-helical stem of hairpin 5′GAAGGAGGAGA- T ₄ -TCTCCTCCTTC (h26) by triple-helix formation (1). In this paper, we report the effect on triplex formation of substituting the cytosine residues of Y11 with 5-methylcytosines (5meY11) . In addition, we have studied the thermodynamics of the interaction between h26 and 5meY11 . The results can be summarised as follows: (i) gel electrophoresis shows that at T = 5°C and pH 5, both Y11 and 5meY11 form DNA triple helices with h26 , whereas at pH 6.8 only the ethylated strand inds to h26 ; (ii) pH-stability curves of the DNA triplexes formed from h26 + Y11 and h26 + 5meY11 show that Y11 and 5meY11 are semi-protonated at pH 5.7 and 6.7, respectively. Thus, it is concluded that cytosine methylation expands the pH range compatible with triplex formation by one pH unit; (iii) as the unmethylated triplex {h26: Y11) , the methylated one (h26: 5meY11) denatures in a biphasic manner, in which the low temperature transition results from the dissociation of 5meY11 from h26 . The Tm of the triplex to h26 plus 5meY11 transition is strongly enhanced (about 10°C) by cytosine methylation. A van 't Hoff analysis of denaturation curves is presented; (iv) DSC experiments show that triplex formation between 5meY11 and h26 is characterized by AH = -237 ±25 kJ/mol and δS = -758 ± 75 J/Kmol, corresponding to an average δH of - 2 1 kJ/mol and δS of - 6 9 J/Kmol per oogsteen base pair; (v) the thermodynamic analysis indicates that the extra stability imparted to the triplex by methylcytosine is enopic in origin