To study nucleosome mobility and positioning, the R3 lac repressor was used with an adenosine triphosphate (ATP)-dependent chromatin assembly system to establish the positioning of five nucleosomes, with one nucleosome located between two R3 lac operators. When R3 protein was dissociated from DNA with isopropyl beta-D-thiogalactopyranoside, the R3-induced nucleosome positions remained unchanged for at least 60 minutes in the absence of ATP but rearranged within 15 minutes in the presence of ATP. These results suggest that nucleosomes are dynamic and mobile rather than static and that a DNA binding factor is continuously required for the maintenance of nucleosome positioning.