Assembly of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex is an essential step for neurotransmitter release in synapses. The presynaptic plasma membrane associated proteins (t-SNAREs), SNAP-25 (synaptosome-associated protein of 25,000 Da) and syntaxin 1A may form an intermediate complex that later binds to vesicle-associated membrane protein 2 (VAMP2). Using spin labeling electron paramagnetic resonance (EPR), we found that the two t-SNARE proteins assemble into a parallel four-helix bundle that consists of two identical syntaxin 1A components and the N-terminal and C-terminal domains of SNAP-25. Although the structure is generally similar to that of the final SNARE complex, the middle region of the helical bundle appears more flexible in the t-SNARE complex. Such flexibility might facilitate interactions between VAMP2 and the t-SNARE complex.