Postnatal brain development continues throughout adolescence into young adulthood. In particular, synapse strengthening and elimination are prominent processes during adolescence. However, molecular data of this relatively late stage of synaptic development are sparse. In this study, we used iTRAQ (isobaric tag for relative and absolute quantification)-based proteomics and electron microscopy to investigate the molecular composition of a synaptic membrane fraction from adolescent postnatal day (P)34 and P44 and adult (P78) rat medial prefrontal cortex. Differential expression of proteins was most prominent between early adolescence and young adulthood (35%, P34–P78), with an over-representation of cell-membrane proteins during adolescent development (between P34 and P44), and synaptic vesicle proteins between late adolescence and young adulthood (P44–P78). Indicative of the critical period of development, we found that, between P34 and P44, a substantial number of proteins was differentially expressed (14%), much more than during the period after adolescence, i.e. between P44 and P78 (5%). A striking observation was the developmental non-stoichiometric regulation of distinct classes of proteins from the synaptic vesicle and the presynaptic release machinery. Electron microscopy demonstrated a small change in the number of docked vesicles between P34 and P44, but not in the total number of synaptic vesicles and in the size of the vesicle cluster. We conclude that the molecular composition of synapses, and more specifically the synaptic release machinery, of the medial prefrontal cortex changes drastically during adolescent development.