The spectra of two double-helical RNA species isolated from virus-like particles found in the mycelium of Penicillium chrysogenum were measured at about 25 degrees C, and at 95 degrees C after denaturation to a single-stranded form, and compared with the spectrum of the equivalent mixture of nucleotides. For both species the difference spectrum (epsilon(95 degrees C)-epsilon(25 degrees C)) when increased by 33% was very similar to the difference spectrum (epsilon(nucleotides)-epsilon(25 degrees C)). In contrast it was found for rat liver DNA that there was no simple relation between (epsilon(95 degrees C)-epsilon(25 degrees C)) and (epsilon(nucleotides)-epsilon(25 degrees C)). These observations were accounted for on the basis of the difference spectra for rA.rU, rG.rC, dA.dT and dG.dC base-pairs derived from model polyribonucleotides and polydeoxyribonucleotides. The difference spectra (epsilon(95 degrees C)-epsilon(25 degrees C)) found for rRNA and those calculated from a combination of the difference spectra for rA.rU and rG.rC base-pairs were in good agreement.