Loading slices of rat barrel cortex with 50 µM BAPTA-AM while recording from pyramidal cells in layer II induces a marked reduction in both the frequency and amplitudes of mEPSCs. These changes are due to a presynaptic action. Blocking the refilling of Ca²⁺ stores with 20 µM cyclopiazonic acid (CPA), a SERCA pump inhibitor, in conjunction with neuronal depolarisation to activate Ca²⁺ stores, results in a similar reduction of mEPSCs to that observed with BAPTA-AM, indicating that the source for intracellular Ca²⁺ is the endoplasmic reticulum. Block or activation of ryanodine receptors by 20 µM ryanodine or 10 mM caffeine, respectively, shows that a significant proportion of mEPSCs are caused by Ca²⁺ release from ryanodine stores. Blocking IP₃ receptors with 14 µM 2-aminoethoxydiphenylborane (2APB) also reduces the frequency and amplitude of mEPSCs, indicating the involvement of IP₃ stores in the generation of mEPSCs. Activation of group I metabotropic receptors with 20 µM (RS)-3,5-dihydroxyphenylglycine (DHPG) results in a significant increase in the frequency of mEPSCs, further supporting the role of IP₃ receptors and indicating a role of group I metabotropic receptors in causing transmitter release. Statistical evidence is presented for Ca²⁺-induced Ca²⁺ release (CICR) from ryanodine stores after the spontaneous opening of IP₃ stores.