To study the dynamics and organization of the DNA within isolated Escherichia coli nucleoids, we track the movement of a specific DNA region. Labeling of such a region is achieved using the Lac-O/Lac-I system. The Lac repressor-GFP fusion protein binds to the DNA section where tandem repeats of the Lac operator are inserted, which allows us to monitor the motion of the DNA. The movement of such a GFP spot is followed at 48 ms temporal resolution during 12 s. The spots are found to diffuse within a confined space, so that the nucleoid appears to behave like a viscoelastic network. The distribution of the &DPRIME; particle&DPRIME; position in time can be fitted to a Gaussian function indicating that the motion of the particle is Brownian. Ail average self-diffusion constant D-s = 0.12 μ m(2) s(-1) is derived via the time auto-correlation functions of the displacement and is compatible with the collective diffusion coefficient measured previously by dynamic light scattering. Restriction of a DNA sequence to a small region of the nucleoid is tentatively related to the existence of so-called supercoiling domains. © 2005 Elsevier Inc. All rights reserved.