The equilibria and kinetics of the intercalation of ethidium bromide (EB) into poly(A).poly(U) have been studied in aqueous solution (pH = 7.0, 1 M NaCl, 25 degreesC) by spectrophotometry, spectrofluorimetry, and stopped-flow techniques. The analysis of the binding isotherms yields the site binding constant K = 2.6 x 10(4) M-1 and the site size n = 2.2 base pairs, thus revealing the occurrence of intercalation according to the excluded site model. The salt dependence of the binding constant indicates that approximately one Na+ counterion is displaced from the polymer by the binding. of one ethidium ion. The kinetic curves are biexponential both in absorbance and in fluorescence. The results suggest the formation of two bound forms according to the reaction scheme D + S (k1)(reversible arrow) DSI and S + DSI (k2)(reversible arrow) DSII + S, where k(1) = 5.9 x 10(4) M-1 s(-1), k(-1) = 8.7 s(-1), k(2) = 1.8 x 10(5) M-1 s(-1), and k(-2)(k-1) = 0.70 x 10(5) M--1(k-2) s(-1). The value of the equilibrium constant K, derived from kinetics as a combination of rate constants, agrees with that derived from equilibria. The behavior of the investigated RNA/ethidium system is compared with that of the DNAs/ethidium systems.