The genomic DNA of Escherichia coli occurs in compact bodies known as nucleoids. Organization and Structure of nucleoids lire poorly understood. Compact, characteristically shaped. nucleoids isolated by the polysine spermidine procedure were visualized by DNA fluorescence microscopy. Treatment with urea or trypsin converted compact nucleoids to partially expanded forms. The transition in urea solutions was accompanied by release of most DNA-associated proteins the transition point between compact and partially expanded forms was not changed by the loss of the proteins nor was it changed in nucleoids isolated from cells after exposure to chloramphenicol or from cells in which Dps, Fis, or H-NS and StpA had been deleted. Partially expanded forms became dispersed upon RNase exposure, indicating a role of RNA in maintaining the partial expansion. Partially expanded forms that had been stripped of most DNA-associated proteins were recompacted by polyethylene glycol 8000, a macromolecular crowding agent, in a cooperative transition. DNA-associated proteins are suggested to have relatively little effect on the phase-like behavior of the cellular nucleoid, Changes in the urea transition indicate that a previously described procedure for compaction of polylysine spermidine nucleoids may have an artifactual basis, and raise questions about reports of repetitive local structures involving the DNA of lysed (C) 2005 Elsevier Inc. All rights reserved.