Bacterial DNA is largely localized in compact bodies known as nucleoids. The structure of the bacterial nucleoid and the forces that maintain its DNA in a highly compact yet accessible form are largely unknown. In the present study, we used urea to cause controlled unfolding of spermidine nucleoids isolated from Escherichia coli to determine factors that are involved in nucleoid compaction. Isolated nucleoids unfolded at similar to 3.2 M urea. Addition of pancreatic RNase reduced the urea concentration for unfolding to similar to 1.8 M urea, indicating a role of RNA in nucleoid compaction. The transitions at similar to 3.2 and similar to 1.8 M urea reflected a RNase-sensitive and a RNase-resistant restraint to unfolding, respectively. Removal of the RNase-sensitive restraint allowed us to test for roles of proteins and supercoiling in nucleoid compaction and structure. The remaining (RNase-resistant) restraints were removed by low NaCl concentrations as well as by urea. To determine if stability would be altered by treatments that caused morphological changes in the nucleoids, transitions were also measured on nucleoids from cells exposed to chloramphenicol; the RNase-sensitive restraint in such nucleoids was stabilized to much higher urea concentrations than that in nucleoids from untreated cells, whereas the RNase-resistant transition appeared unchanged.