Formation of complexes between soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins on opposing membranes is the minimal requirement for intracellular membrane fusion. The SNARE, syntaxin 2, is found on the sperm plasma membrane and a second SNARE, vesicle associated membrane protein 2 (VAMP2, also known as synaptobrevin 2, SYB2), is on the apposing outer acrosomal membrane. During the acrosome reaction, the outer acrosomal membrane fuses at hundreds of points with the plasma membrane. We hypothesized that syntaxin 2 and VAMP2 redistribute within their respective membranes prior to the acrosome reaction to form trans-SNARE complexes and promote membrane fusion. Immunofluorescence and superresolution structured illumination microscopy were used to localize syntaxin 2 and VAMP2 in mouse sperm during capacitation. Initially, syntaxin 2 was found in puncta throughout the acrosomal region. At 60 and 120 min of capacitation, syntaxin 2 was localized in puncta primarily in the apical ridge. Although deletion of bicarbonate during incubation had no effect, syntaxin 2 puncta were relocated in the restricted region in less than 20% of sperm incubated without albumin. In contrast, VAMP2 was already found in puncta within the apical ridge prior to capacitation. The puncta containing syntaxin 2 and VAMP2 did not precisely co-localize at 0 or 60 min of capacitation time. In summary, syntaxin 2 shifted its location to the apical ridge on the plasma membrane during capacitation in an albumin-dependent manner but VAMP2 was already localized to the apical ridge. Puncta containing VAMP2 did not co-localize with those containing syntaxin 2 during capacitation; therefore, formation of trans-SNARE complexes containing these SNAREs does not occur until after capacitation, immediately prior to acrosomal exocytosis.