N-Ethylmaleimide-sensitive factor (NSF) is an ATPase involved in many membrane fusion events within the exocytic and endocytotic pathways. In the present study we showed that NSF is associated with the nuclear envelope. Golgi-associated NSF was released from membranes upon incubation with Mg2+-ATP, reflecting the disassembly of a complex consisting of NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs), In contrast nuclear envelope-associated NSF in interphase cells was not released by the same treatment. During mitosis, however, it was released from nuclear membranes by Mg2+-ATP. These results suggest that the binding mode of nuclear membrane-associated NSF changes during the cell cycle,