Fis, the most abundant DNA-binding protein in Escherichia coli during rapid growth, has been suspected to play an important role in defining nucleoid structure. Using bulk-phase and single-DNA molecule experiments, we analyze the structural consequences of non-specific binding by Fis to DNA. Fis binds DNA in a largely sequence-neutral fashion at nanomolar concentrations, resulting in mild compaction under applied force due to DNA bending. With increasing concentration, Fis first coats DNA to form an ordered array with one Fis dimer bound per 21 bp and then abruptly shifts to forming a higher-order Fis-DNA filament, referred to as a low-mobility complex (LMC). The LMC initially contains two Fis dimers per 21 bp of DNA, but additional Fis dimers assemble into the LMC as the concentration is increased further. These complexes, formed at or above 1 microM Fis, are able to collapse large DNA molecules via stabilization of DNA loops. The opening and closing of loops on single DNA molecules can be followed in real time as abrupt jumps in DNA extension. Formation of loop-stabilizing complexes is sensitive to high ionic strength, even under conditions where DNA bending-compaction is unaltered. Analyses of mutants indicate that Fis-mediated DNA looping does not involve tertiary or quaternary changes in the Fis dimer structure but that a number of surface-exposed residues located both within and outside the helix-turn-helix DNA-binding region are critical. These results suggest that Fis may play a role in vivo as a domain barrier element by organizing DNA loops within the E. coli chromosome.