The effects of Ca²⁺, Sr²⁺ and Ba²⁺ on spontaneous and evoked glycinergic inhibitory postsynaptic currents (mIPSCs and eIPSCs) were investigated using the 'synaptic bouton' preparation of rat spinal neurons and conventional whole-cell recording under voltage-clamp conditions. In response to application of Ca²⁺-free solution, the frequency of mIPSC initially rapidly decreased to 40 ~ 50 % of control followed by a gradual further decline in mIPSC frequency to ~ 30 % of control. Once mIPSC frequency had significantly declined in Ca²⁺-free solution, application of Ca²⁺, Sr²⁺ or Ba²⁺ increased mIPSC frequency. The rank order of effect in restoring mIPSCs was Ba²⁺ >> Ca²⁺ > Sr²⁺. Moreover, the application of excess external [K⁺]_o solution (30 mM) containing Sr²⁺ or Ba²⁺ after 2 hrs in Ca²⁺-free solution also increased mIPSC frequency in the order Sr²⁺ Ba²⁺ > Ca²⁺. The mean mIPSC amplitude was not affected at all. In contrast, eIPSCs produced by focal stimulation of single boutons were completely abolished in Ca²⁺-free solution or when Ca²⁺ was replaced by Sr²⁺ or Ba²⁺ (2 mM each). However eIPSCs were restored in increased concentrations of Sr²⁺ or Ba²⁺ (5 mM each). The results show that these divalent cations affect mIPSC and eIPSCs differently, and indicate that the mechanisms underlying transmitter release that generates eIPSCs and mIPSC in presynaptic nerve terminals are different. The different mechanisms might be explained by the different sensitivity of synaptotagmin isoforms to Ca²⁺, Sr²⁺ and Ba²⁺.