A possible switch between two conformations, previously observed in an enzymatically cleaved fragment of E. coli 5S ribosomal RNA (a Gram-negative bacterium) containing helices II and III, has been examined by means of proton nuclear magnetic resonance spectroscopy (10-15 ppm) as a function of [Mg2+] and temperature for an RNase-T1 digested fragment of Bacillus megaterium 5S rRNA (a Gram-positive bacterium) containing the same helices II and III. The conformational changes induced in the fragment are not accompanied by breakage of some base-pairs and formation of others, but rather consist simply of tightening or loosening of helices with retention of existing base-pairs. Helix III is found to be more flexible than helix II. Finally, the loop conformation is conserved over a wide range of Mg2+ concentration, suggesting that the loop may serve an important role in the biological function of 5S rRNA in ribosomes.