Acrosomal exocytosis in mammalian sperm is a regulated secretion with unusual characteristics. One of its most striking features is the loss of the outer acrosomal membrane and the overlying plasma membrane as hybrid vesicles. We have reported previously in human sperm that by preventing the release of calcium from the acrosome, the exocytic process can be arrested at a stage where SNARE proteins are assembled in loose trans complexes. Transmission electron micrographs of sperm at this stage showed that the acrosomes were profusely swollen, with deep invaginations of the outer acrosomal membrane. The protruding edges of these invaginations were tightly apposed (i.e., docked) to the plasma membrane. Docking was prevented when streptolysin O-permeabilized sperm were stimulated in the presence of tetanus toxin or botulinum neurotoxin C, two SNARE-specific proteases. We propose that SNAREs present in the plasma membrane interact with SNAREs in the protruding edge of cup-shaped invaginations of the outer acrosomal membrane to form trans complexes. Fusion pore opening and expansion in this ring of apposed membranes would generate the hybrid vesicles that are released during the acrosome reaction.