We used actin tagged with enhanced green fluorescent protein (EGFP-actin) to characterize the distribution and dynamics of actin in living presynaptic terminals in rat CNS neurons. Actin was preferentially concentrated around--and appeared to surround--the presynaptic vesicle cluster. In resting terminals, approximately 30% of actin was found to be in a polymerized but dynamic state, with a remodeling time scale of approximately 20 s. During electrical activity, actin was further polymerized and recruited from nearby axonal regions to the regions surrounding vesicles. Treatment of terminals with the actin monomer-sequestering agent latrunculin-A completely dispersed the actin network and abolished activity-dependent actin dynamics. We used a variety of methods to examine the role of actin in the presynaptic vesicle cycle. These data rule out a propulsive role for actin, either in maintaining the vesicle cluster or in guiding vesicle recycling. Instead, we propose that actin acts as a scaffolding system for regulatory molecules in the nerve terminal.