Following up our studies on the differential expression of presynaptic proteins in glutamatergic and GABAergic axon terminals (Bragina et al., 2007; 2010; 2012), we performed triple labeling studied on the extent of co-localization between synaptotagmin (SYT) 1 and 2, in VGLUT1-positive (+) and VGLUT2+ axon terminals, of Rab3a and c in VGLUT1+, VGLUT2+ and VGAT+ terminals and of syntaxin (STX) 1A and B in VGLUT1+ and VGLUT2+ puncta. Co-localization studies showed that ~75 % of glutamatergic terminals that expressed SYT2 expressed also SYT1, ~4 % expressed only SYT2, ~57 % expressed only SYT1 and 27% none. Analysis of STX isoforms provided similar results, for ~ 50% of VGLUT1+ axon terminals expressed both isoforms, whereas the remaining terminals expressed neither STX1A nor STX1B. As for expression of Rab3 isoforms, results showed that: 1) ~ 70% of VGLUT1+ axon terminals expressing Rab3c co-express Rab3a, ~58 % expressed only Rab3a, ~3,5 expressed only Rab3c and ~ 30 % none; 2) ~55 % of VGLUT2 (+) terminals expressing Rab3c expressed also Rab3a, ~ 20 % expressed only Rab3a and ~ 10 % expressed only Rab3c and ~ 57% none; and 3) ~ 45 % of VGAT+ axon terminals expressing Rab3c expressed also Rab3a, ~ 30 % expressed only Rab3a or Rab3c. In addition, immunoisolation experiments show that synaptic vesicles can express both isoforms of SYT. Thus, triple-labeling studies showed that the degree of heterogeneity is higher than previously depicted by double-labeling studies.