Cav2.1 channels are critically important for fast synaptic transmission at central synapses. The two Cav2.1 isoforms Cav2.1[EFa] and Cav2.1[EFb] are generated by alternative splicing of two mutually exclusive exons, 37a and 37b. Both isoforms are expressed throughout the adult brain, whereas Cav2.1[EFb] predominates at early stages of development. Cav2.1[EFa] supports synchronous release and short-term synaptic depression, whereas Cav2.1[EFb] boosts asynchronous release and short-term facilitation. We investigated whether the two isoforms were distinctly located at excitatory nerve terminals relative to components of the vesicle release complex. We infected primary cortical neurons with lentiviruses expressing Cav2.1[EFa] or Cav2.1[EFb] and analyzed their expression at 18-20 DIV using super-resolution structured illumination microscopy (SIM). Images quantification showed that the exogenous Cav2.1[EFa] and Cav2.1[EFb] did not alter the size of Cav2.1 puncta, nor structure or number of synaptic boutons. Cav2.1[EFa] increased the amount of co-localization of Cav2.1 with Basoon and vGlut1 puncta relative to non-infected neurons, while Cav2.1[EFb] had no effect. Likewise, Munc-13, a key component of the vesicle release machinery, exhibited a higher degree of overlap with Cav2.1[EFa] compared to Cav2.1[EFb]. These results indicate that the Cav2.1 [EFa] splice variant is tightly co-localized with the active zone, supporting synchronous release of synaptic vesicles. On the contrary, Cav2.1[EFb] is loosely coupled with Ca2+ sensors, boosting residual Ca2+ and short-term facilitation.