Synaptic vesicle (SV) size is one parameter that controls the amount of neurotransmitter released from individual SVs and, therefore, is fundamental to our understanding of synaptic function. The recently discovered variability of mean SV size among excitatory hippocampal synapses—if actively regulated—is a potential mechanism for the regulation of transmitter release. Here, we investigated which parameters influence mean SV size. First, we revealed that synapse-to-synapse variability of SV size is a general phenomenon in several species and brain regions. In addition, we determined the relationship between mean SV size and synaptic morphology. In three-dimensional reconstructions from serial ultrathin sections, we found that SV size did not correlate with the area of the postsynaptic density (a measure for synaptic size and synaptic cleft volume) nor with the total number of SVs within a bouton or bouton volume. We tested the long-held hypothesis that a change in osmotic pressure (potentially caused by a change in neurotransmitter concentration) affects SV size. When we reduced the osmotic pressure, SVs became significantly smaller; however, an increase in osmotic pressure had no effect on SV size. Furthermore, we found that SV size does not adapt to chronic changes in activity and that the SV cycle is capable of providing constant SV size during long-lasting, high-frequency stimulation. J. Comp. Neurol. 514:343–352, 2009. © 2009 Wiley-Liss, Inc.