AbstractSecretory vesicle swelling is central to cell secretion, but the underlying mechanism of vesicle swelling, particularly synaptic vesicles, is not completely understood. The Gαi3‐PLA2‐mediated involvement of water channel AQP‐1 in the regulation of secretory vesicle swelling in exocrine pancreas and the Gαo‐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‐Gαo‐mediated synaptic vesicle swelling. Results from this study demonstrate that the GTP‐Gαo‐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‐Gαo‐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 Gαo‐stimulated swelling of synaptic vesicles. Vesicle acidification is therefore a prerequisite for AQP‐6‐mediated gating of water into synaptic vesicles. © 2009 Wiley‐Liss, Inc.