To gain insight into the role of F-actin in the organization of synaptic vesicles at release sites, we examined the synaptic distribution of F-actin by using a unique synaptic preparation of frog target-deprived nerve terminals. In this preparation, imaging of the synaptic site was unobstructed by the muscle fiber cytoskeleton, allowing for the examination of hundreds of synaptic sites in their entirety in whole mounts. At target-deprived synaptic sites F-actin was distributed in a ladder-like pattern and was colocalized with β-fodrin. Surprisingly, F-actin stain, which we localized to the nerve terminal itself, did not overlap a synaptic vesicle marker, suggesting that it was concentrated in nonrelease domains of nerve terminals between clusters of synaptic vesicles. These findings suggest that the majority of the presynaptic F-actin is not involved in tethering synaptic vesicles. Instead, the strategic presynaptic positioning of this cytoskeletal meshwork in nonrelease domains of the nerve terminal suggests alternate functions such as restricting synaptic vesicles to release domains, recycling synaptic vesicles, or stabilizing the nerve terminal.