Striatal cholinergic interneurons (CINs) use both acetylcholine (ACh) and glutamate (Glut) to regulate the striatal network. CINs express vesicular transporters for acetylcholine (VAChT) and glutamate (VGLUT3) with a high degree of coexpression at axon terminals. We have used super resolution STimulated Emission Depletion microscopy to characterize the distribution of VAChT and VGLUT3 in synaptic vesicles (SVs) from CINs. Analysis of the relative distance distribution revealed that 33% of cholinergic SVs contain both VAChT and VGLUT3. In addition, 40% of SVsfrom CINS express only VAChT whereas 27% contain VGLUT3. It can thus be proposed that CINs terminals have the potential to release simultaneously or independently ACh and/or Glut. Our results advocate for an unexpected level of anatomical and functional heterogeneity of SVs in striatal cholinergic varicosities.