The infrared spectra of ribosomal 5S RNAs of Escherichia coli and rat liver were measured under conditions where the structure of 5S RNA is preserved. The comparison of the spectra shows significant spectral differences for the U residues which resemble in their characteristics the infrared spectra of polyuridylic acid in its base paired state. This strongly indicates the presence of U.U mispairs in rat liver 5S RNA. Juxtaposed U residues at positions U80-U96 and U73-U102 are potential candidates for U.U mispairs in rat liver 5S RNA.