Midbrain dopamine (DA) neurons comprise a heterogeneous population of cells. For instance, some DA neurons express the vesicular glutamate transporter VGLUT2 allowing these cells to co-release DA and glutamate. Additionally, GABA may be co-released from DA neurons. However, most cells do not express the canonical machinery to synthesize GABA or the vesicular GABA transporter VGAT. Instead, GABA seems to be taken up into DA neurons by a plasmalemmal GABA transporter (GAT1) and stored in synaptic vesicles via the vesicular monoamine transporter VMAT2. Yet, it remains unclear whether GABA indeed interacts with VMAT2. Here, we used radiotracer flux measurements in VMAT2 expressing HEK-293 cells and synaptic vesicles from male and female mice to determine whether GABA qualifies as substrate at VMAT2. We found that GABA reduced uptake of VMAT2 substrates in mouse synaptic vesicle preparations from striatum and cerebellum at millimolar concentrations but had no effect in VMAT2-expressing HEK-293 cells. Interestingly, while the closely related amino acid glycine did not affect substrate uptake at VMAT2 in mouse synaptic vesicles, the amino sulfonic acid taurine reduced uptake similar to GABA. Lastly, we discovered that the majority of mouse and human midbrain DA neurons in the substantia nigra of either sex expressed VMAT2 and GAT1 suggesting that most of them could be capable of co-releasing DA and GABA. Together, our findings suggest that GABA is a low-affinity substrate at VMAT2 with potential implications for basal ganglia physiology and disease.