The complex formed between acridine orange (AO) and polycytidylic acid (poly(C)) was studied by spectrophotometry and spectrofluorometry. The complex was characterized by its stoichiometry, structure, and the thermodynamic parameters of its formation. The results are in agreement with an external aggregation of the protonated dye along the negatively charged poly(C) chain and indicate that approximately two AO molecules are bound per nucleotide unit of poly(C). The kinetics of the reaction between this complex and a Pd(II) complex was studied by the stopped-flow method. The addition of (dien)Pd(II) to the AO–poly(C) complex leads to the dissociation of the latter, due to fixation of the Pd(II) complex to the N3 site of the cytosine base of poly(C). The rate constant for the AO liberation, extrapolated at zero AO concentration, corresponds to the rate constant of Pd(II) fixation on poly(C). This indicates that AO can be used as an indicator for this reaction and allows kinetic studies at very low concentrations (≤ 5 × 10−6 M).