Although it is established that presynaptic actin participates in synaptic vesicle recycling at several synapses, the earliest stages at which actin polymerization is employed during this process are still unclear. To address this, we prevented actin polymerization at lamprey synapses by applying latrunculin B or swinholide A. Latrunculin and swinholide depolymerize actin by sequestering actin monomers and, in addition, swinholide can sever existing actin filaments. When injected into individual presynaptic axons of the intact spinal cord, fluorescently labeled monomeric actin rapidly incorporated in a calcium-dependent manner into a stable, filamentous actin network concentrated at endocytic zones. This pool of actin was disrupted completely by latrunculin. At stimulated synapses, specific disruption of actin polymerization with latrunculin and swinholide induced a selective increase in unconstricted clathrin-coated pits and, in the case of swinholide, an additional increase in the size of plasma membrane evaginations. These results indicate that actin polymerization participates initially in the maturation of clathrin-coated pits during early stages of synaptic vesicle recycling.