The stabilization of poly(dA).poly(dT) tripler, a 22-base DNA tripler, and poly(rA).2poly(rU) triple helix by neomycin is reported. The: melting temperatures, the association and dissociation kinetic parameters, and activation energies (E-on and E-off) for the poly(dA).2poly(dT) tripler in the presence of aminoglycosides and other tripler binding ligands were determined by UV thermal analysis. Our results indicate that: (i) neomycin stabilizes DNA triple helices, and the double helical structures composed of poly(dA) poly(dT) are virtually unaffected. (ii) Neomycin is the most active and tripler-selective stabilization agent among all aminoglycosides, previously studied minor groove binders, and polycations. Its selectivity (DeltaT(m3 -->2) vs DeltaT(m2 -->1)) exceeds most intercalating drugs that bind to triple helices. (iii) Neomycin selectively stabilizes DeltaT(m3 -->2) for a mixed 22-base DNA tripler containing C and T bases in the pyrimidine strand. (iv) The rate constants of formation of tripler (k(on)) are significantly enhanced upon increasing molar ratios of neomycin, making tripler association rates closer to duplex association rates. (v) E,, values become more negative upon increasing concentration of aminoglycosides (paromomycin and neomycin). E-off values do not show any change for most aminoglycosides except neomycin. (vi) Aminoglycosides can effectively stabilize RNA {poly(rA).2poly(rU)} tripler, with neomycin being one of the most active ligands discovered to date (second only to ellipticine). (vii) The stabilization effect of aminoglycosides on triple helices is parallel to their toxic behavior, suggesting a possible role of intramolecular triple helix (H-DNA) stabilization by the aminoglycosides.