Whether actin has a role in endocytosis in mammalian cells is unclear. Wu et al. showed that, in mice, knockout of the β-actin gene or γ-actin gene impaired various types of endocytosis in calyx-type and hippocampal synapses. The deficits observed in hippocampal boutons could be rescued by the expression of wild-type actin isoforms but not by polymerization-deficient mutant actin. Moreover, knockout of the β-actin gene led to a reduction in the number of membranous pits in such boutons. These data suggest that polymerized actin is crucial for endocytosis at different synapses.