Presynaptic structural modifications are thought to accompany activity-dependent synaptic plasticity and learning. This may involve the conversion of nonfunctional synapses into active ones or the generation of entirely new synapses. Here, using an in vitro neural analog of classical conditioning, we investigated presynaptic structural changes restricted to auditory nerve synapses that convey the conditioned stimulus (CS) by tract tracing using fluorescent tracers combined with immunostaining for the synaptic vesicle-associated protein synaptophysin. The results show that the size of presynaptic auditory boutons increased and the area and fluorescence intensity of punctate staining for synaptophysin were enhanced after conditioning. This occurred only for auditory nerve boutons apposed to the dendrites but not the somata of abducens motor neurons. Conditioning increased the percentage of boutons that were immunopositive for synaptophysin and enhanced the number of synaptophysin puncta they contained. Pretreatment with antibodies against brain-derived neurotrophic factor (BDNF) inhibited these conditioning-induced structural changes. There was also a net increase in the number of boutons apposed to abducens motor neurons after conditioning or BDNF treatment. These data indicate that the rapid enrichment of presynaptic boutons with proteins required for neurotransmitter recycling and release occurs during classical conditioning and that these processes are mediated by BDNF. © 2011 IBRO.