gamma-Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (gamma-SNAP) is capable of stabilizing a 20 S complex consisting of NSF, alpha-SNAP, and SNAP receptors (SNAREs), but its function in vesicular transport is not fully understood. Our two-hybrid analysis revealed that gamma-SNAP, unlike alpha-SNAP, interacts directly with NSF, as well as Gaf-1/Rip11, but not with SNAREs. Gaf-1/Rip11 is a gamma-SNAP-associated factor that belongs to the Rab11-interacting protein family. To gain insight into the molecular basis for the interactions of gamma-SNAP with NSF and Gaf-1/Rip11, we determined the regions of the three proteins involved in protein-protein interactions. gamma-SNAP bound to NSF via its extreme C-terminal region, and the full-length NSF was needed to interact with gamma-SNAP. Both the N-terminal and C-terminal regions of gamma-SNAP were required for the binding to Gaf-1/Rip11. Gaf-1/Rip11 bound to gamma-SNAP via its C-terminal domain comprising a putative coiled-coil region. Although the C-terminal domain of Gaf-1/Rip11 also interacts with Rab11, the binding of gamma-SNAP and Rab11 to Gaf-1/Rip11 was not mutually exclusive. Rather, Gaf-1/Rip11 was capable of serving a link between gamma-SNAP and Rab11. A complex comprising gamma-SNAP and Gaf-1/Rip11 was disassembled in a process coupled to NSF-mediated ATP hydrolysis, suggesting that the interaction between gamma-SNAP and Gaf-1/Rip11 is of functional significance.