The release of transmitter at neuromuscular junctions (NMJ) of the opener muscle in crayfish is quantal in nature. This NMJ offers the advantage of being able to record quantal events at specific visually identified release sites, thus allowing measurement of the physiological parameters of vesicle release and its response to be directly correlated with synaptic structure. These experiments take advantage of areas between the varicosities on the nerve terminal that we define as “stems.” Stems were chosen as the region to study because of their low synaptic output due to fewer synaptic sites. Through 3D reconstruction from hundreds of serial sections, obtained by transmission electron microscopy (TEM), at a site in which focal macropatch recordings were obtained, the number of synapses and AZs are revealed. Thus, physiological profiles with various stimulation conditions can be assessed in regards to direct synaptic structure. Here, we used the properties of the quantal shape to determine if distinct subsets of quantal signatures existed and if differences in the distributions are present depending on the frequency of stimulation. Such a quantal signature could come about by parameters of area, rise time, peak amplitude, latency, and tau decay. In this study, it is shown that even at defined sites on the stem, with few active zones, synaptic transmission is still complex and the quantal responses appear to be variable even for a given synapse over time. In this study, we could not identify a quantal signature for the conditions utilized. Synapse, 2011. © 2010 Wiley-Liss, Inc.