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The 5S RNAs from Bacillus stearothermophilus and Saccharomyces cerevisiae were probed by nucleotide-specific reagents, with a view to compare and contrast their higher order structures. The progressive unfolding of the RNAs during heating, in the presence and absence of magnesium, was monitored. Evidence was provided for the double-helical segments which occur in the secondary structural models of both RNAs. The results also placed constraints on the possible structuring of the remainder of the RNA and yielded some insight into ways of folding up the molecule. Together with the data from our earlier studies, employing ribonucleases, these results provide a detailed picture of the structuring and topography of the 5S RNAs. The main structural differences between the eubacterial and eukaryotic RNAs occur throughout the loop D/helix IV/loop E/helix V arm; in particular strong evidence is provided for loop D of the eukaryotic RNA being involved in a tertiary interaction.