Synaptophysin is an abundant synaptic vesicle protein without a definite synaptic function. Here, we examined a role for synaptophysin in synapse formation in mixed genotype micro-island cultures of wild-type and synaptophysin-mutant hippocampal neurons. We show that synaptophysin-mutant synapses are poor donors of presynaptic terminals in the presence of competing wild-type inputs. In homogenotypic cultures, however, mutant neurons display no apparent deficits in synapse formation compared with wild-type neurons. The reduced extent of synaptophysin-mutant synapse formation relative to wild-type synapses in mixed genotype cultures is attenuated by blockers of synaptic transmission. Our findings indicate that synaptophysin plays a previously unsuspected role in regulating activity-dependent synapse formation.