The kinetics and equilibria of the interaction of ethidium bromide (EB) with the triple-stranded RNA, poly( rA)center dot 2poly(rU), have been investigated by stopped-flow, absorption, fluorescence, and circular dichroism methods; to properly assess the effect of the third strand on the polymer molar properties, molar volumes, adiabatic compressibilities, and heats of melting have also been measured for both poly( rA), 2poly( rU) and poly( rA), poly( rU). The melting experiments reveal that ethidium tends to destabilize the triplex, whereas it stabilizes the duplex; however, the triplex/ethidium system in 0.1 M NaCl is stable below 37 C. The static titrations reveal that one ethidium ion binds every three base triplets of the polymer; on the basis of the excluded-site model, this feature suggests intercalation, as in the duplex, but the binding affinity for the triplex is weaker compared to that for the duplex. The kinetic experiments displayed a two-phase behavior, which was rationalized assuming the sequence D + S reversible arrow DSI, DSI + S reversible arrow DSII + S (D = drug, S = site), the second step involving direct transfer of the drug between strands. Comparison with the duplex/EB system reveals that the additional strand of poly( U), present in the triplex, hinders the formation of the intermediate complex DSI, while stabilizing the structure of the final DSII complex by hampering the partial slipping out of the dye from the triplex cavity.