Complementary short-strand DNA homooligomers and methylthiourea linked homonucleosides associate and form triplexes in solution. The melting temperatures, T-m, and the association and dissociation kinetic and thermodynamic parameters were determined by UV thermal analysis for the triplexes of short-strand DNA homooligomers (1-A(20), 2-CA(4)CA(4)CA(4)C, 3-C(2)A(3)C(2)A(3)C(2)A(3)C(2), 4-C(3)A(2)C(3)A(2)C(3)A(2)C(2), and 5-CACACACACACACACACACA) with the S-methylthiourea linked nucleoside {5'-NH3+-d(Tmt)(4)-T-OH (DNmt(5))}, Circular dichroism studies show evidence of triple-helical association dependent on the mismatch content of the target homooligomer. The melting and cooling curves exhibit hysteresis behavior in the temperature range of 10-95 degrees C at 0.13 deg/min thermal rate. From these curves the rate constants for association (k(on)) and dissociation (k(off)) were obtained. T-m decreases drastically with increase in mismatch of the target oligomer. The rate constants k(on) and k(off) at a given temperature (288-310 K) are dependent on the C/A ratio. The free energies of tripler formation (Delta G degrees) become appreciably less negative as the ratio of C to A increases. Thymidyl DNmt is shown to bind to DNA oligomers with high fidelity under physiological conditions.