The tertiary structure of the eukaryotic ribosomal 5 S RNA was examined using ethylnitrosourea reactivity as a probe for phosphodiester bonds. In three different 5 S RNAs of diverse origin the reactivity was restricted to the same three regions of the sequence, corresponding to residues G 99-A 101, A 88-G 89, and G 75 in the rat 5 S RNA molecule. All of these restricted residues are in highly conserved sequences; five of the residues (G 75, G 89, G 99, A 100, and A 101 in the rat) are present in all 5 S RNAs. The results indicate that, as has been suggested for the secondary structure, the tertiary structure of the 5 S RNA is also highly conserved. Based on these data, a working model for the tertiary structure is suggested which is consistent with previous studies on the structure of the free RNA and 5 S RNA-protein complexes.