What determines where synapses will form along an axon or how proteins are deposited at nascent synapses remains unknown. Here, we show that the initial formation of presynaptic terminals occurs preferentially at predefined sites within the axons of cortical neurons. Time-lapse imaging of synaptic vesicle protein transport vesicles (STVs) indicates that STVs pause repeatedly at these sites, even in the absence of neuronal or glial contact. Contact with a neuroligin-expressing non-neuronal cell induces formation of presynaptic terminals specifically at these STV pause sites. Remarkably, formation of stable contacts with dendritic filopodia also occurs selectively at STV pause sites. Although it is not yet known which molecules comprise the predefined sites, STV pausing is regulated by cues that affect synaptogenesis. Overall, these data are consistent with the hypothesis that regulation of STV pausing might be an important mechanism for accumulation of presynaptic proteins at nascent synapses and support a new model in which many en passant synapses form specifically at predefined sites in young axons.