Studies demonstrate that cholesterol plays a critical role in the regulation of neurotransmitter release and that secretory vesicle swelling is a requirement for the regulated expulsion of intravesicular contents during cell secretion. In view of this, the involvement of cholesterol in synaptic vesicle swelling was hypothesized and tested in the present study, using isolated synaptic vesicles from rat brain and the determination of their swelling competency in the presence and absence of cholesterol. The involvement of the water channel aquaporin-6 (AQP-6) and proton pump vH(+)-ATPase in GTP-G(alpha o)-mediated synaptic vesicle swelling has been reported previously. Mastoparan, the amphiphilic tetradecapeptide from wasp venom, known to activate the GTPase activity of G(alpha o/i) proteins, stimulates synaptic vesicle swelling in the presence of GTP. In the current study, using nanometer-scale precision measurements of isolated synaptic vesicles, we report for the first time that depletion of cholesterol from synaptic vesicle membrane results in a significant loss of GTP-mastoparan-stimulable synaptic vesicle swelling. In contrast, incorporation of cholesterol into the synaptic vesicle membrane potentiates GTP-mastoparan-stimulable vesicle swelling. Our study further demonstrates that this effect of cholesterol is due, in part, to its involvement in the interactions between AQP-6, vH(+)-ATPase and the GTP-binding G(alpha o) protein at the synaptic vesicle membrane.