Abstract The addition of urea to 50-S ribosomal subunits caused a lowering of the sedimentation coefficient to 32–38 S by a one-step co-operative process. This was interpreted as an unfolding of the native ribosome caused by the disruption of hydrophobic bonds between ribosomal proteins. In many cases slow-moving components were evident in the ultracentrifuge which were probably dissociated protein. Treatment of 50-S ribosomes with formaldehyde prevented the dissociation of the protein from RNA on subsequent reaction with urea. The ribosomes appeared to unfold to give slower-sedimenting species but the nature of the unfolding was different from that of untreated ribosomes. Thus the ribosomes were more sensitive to reaction with urea and the unfolding was no longer highly co-operative. The addition of varying amounts of EDTA to formaldehyde-treated ribosomes produced a single unfolded component with a sedimentation coefficient of 34–38 S. Unlike untreated ribosomes no further unfolding occurred, either with time or with a large excess of EDTA. No loss of secondary structure in the RNA occurred when native or formaldehyde-treated ribosomes were reacted with urea or EDTA at room temperature. The melting temperatures decreased in urea and EDTA and the melting curves became less co-operative. The melting profiles of unfolded ribosomes in urea and EDTA were similar to RNA in the same solvent and showed that in the unfolded form the protein had little effect on the thermal denaturation properties of the RNA. It is concluded that in the unfolded ribosomes, characterised by a sedimentation coefficient of 30–35 S, the major bonds involved in the maintenance of ribosomal tertiary structure have been broken. These bonds involve interactions between the proteins of the ribosome.