The photodynamic inactivation of amino acid (phenylalanine) accepting ability of transfer RNA (tRNA) sensitized with acridine orange (AO) was investigated placing a special emphasis on the dye binding to nucleic acid. The dye binding to tRNA was found to be a prerequisite for the photodynamic inactivation of tRNA. The photodynamic efficiencies of AO were compared between two types of tRNA-AO complexes. Based on the calculated quantum yields for the inactivation, the intercalated (monomer) dye was about four times more efficient than the bound dye aggregate. Increasing the number of bound dyes in intercalated form from one to five showed only small variations of the quantum yields, implying that the binding sites in the tRNA is not important insofar as they receives AO in the intercalated form.