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A stunning example of synaptic diversity is the postsynaptic target cell-type-dependent difference in synaptic efficacy in cortical networks. Here, we show that CA1 pyramidal cell (PC) to fast spiking interneuron (FSIN) connections have 10-fold larger release probability (P v) than those on oriens lacunosum-moleculare (O-LM) interneurons. Freeze-fracture immunolabeling revealed that different nano-topologies and coupling distances between Ca2+ channels and release sites (RSs) are not responsible for the distinct P v. Although [Ca2+] transients are 40% larger in FSINs innervating boutons, when [Ca2+] entry is matched in the two bouton populations, EPSCs in O-LM cells are still 7-fold smaller. However, application of a phorbol ester analog resulted in a ∼2.5-fold larger augmentation at PC – O-LM compared to PC – FSIN synapses, suggesting incomplete docking or priming of vesicles. Similar densities of docked vesicles rule out distinct RS occupancies and demonstrate that incompletely primed, but docked, vesicles limit the output of PC – O-LM synapses. [Display omitted] • Probability of release from pyramidal cells differs depending on the target cell type • Replica immunolabeling reveals similar vesicle - Ca2+ channel coupling distances • At low P v synapses, PDBU is more effective in increasing P v than elevating [Ca2+] • Similar docked vesicle densities suggest distinct priming states underlie different P v Aldahabi et al. demonstrate that the target cell-type-dependent difference in release probability from hippocampal pyramidal cell axons is not the consequence of distinct coupling distances or nano-topologies of docked vesicles and Ca2+ channels. Instead, their data reveal robust differences in the priming state of docked vesicles. [ABSTRACT FROM AUTHOR]