Secretory vesicle swelling is central to cell secretion, but the underlying mechanism of vesicle swelling, particularly synaptic vesicles, is not completely understood. The G(alphai3)-PLA2-mediated involvement of water channel AQP-1 in the regulation of secretory vesicle swelling in exocrine pancreas and the G(alphao)-mediated AQP-6 involvement in synaptic vesicle swelling in neurons have previously been reported. Furthermore, the role of vH(+)-ATPase in neurotransmitter transport into synaptic vesicles has also been shown. Using nanometer-scale precision measurements of isolated synaptic vesicles, the present study reports for the first time the involvement of vH(+)-ATPase in GTP-G(alphao)-mediated synaptic vesicle swelling. Results from this study demonstrate that the GTP-G(alphao)-mediated vesicle swelling is vH(+)-ATPase dependent and pH sensitive. Zeta potential measurements of isolated synaptic vesicles further demonstrate a bafilomycin-sensitive vesicle acidification, following the GTP-G(alphao)-induced swelling stimulus. Water channels are bidirectional and the vH(+)-ATPase inhibitor bafilomycin decreases both the volume of isolated synaptic vesicles and GTP-mastoparan stimulated swelling, suggesting that vH(+)-ATPase is upstream of AQP-6, in the pathway leading from G(alphao)-stimulated swelling of synaptic vesicles. Vesicle acidification is therefore a prerequisite for AQP-6-mediated gating of water into synaptic vesicles.