Short-term synaptic plasticity changes the reliability of transmission during repetitive activation and allows different neuronal ensembles to encode distinct features of action potential trains. Identifying the mechanisms and the locus of expression of such plasticity is essential for understanding neuronal information processing. To determine the quantal parameters and the locus of alterations during short-term plasticity of cortical glutamatergic synapses, EPSCs were evoked in hippocampal oriens-alveus interneurons by CA1 pyramidal cells. The robust short-term facilitation of this connection allowed us to examine the transmission under functionally relevant but widely different release probability (P(r)) conditions. Paired whole-cell recordings permitted the functional and post hoc morphological characterization of the synapses. To determine the quantal size (q), the P(r), and the number of functional release sites (N(F)), two independent quantal analysis methods were used. Light and electron microscopy were performed to identify the number of synaptic junctions (N(EM)) between the recorded cells. The mean number of functional release sites (N(F(f)) = 2.9 +/- 0.4; n = 8) as inferred from a simple binomial model with no quantal variance agreed well with the mean of N(EM) (2.8 +/- 0.8; n = 6), but N(F(f)) never matched N(EM) when they were compared in individual pairs; however, including quantal variance in the model improved the functional prediction of the structural data. Furthermore, an increased P(r) (4.8 +/- 0.8-fold) fully accounted for the marked short-term facilitation of EPSCs (5.0 +/- 0.7-fold), and q was independent of P(r). Our results are consistent with the "one-release site, one-vesicle" hypothesis.