Delayed neuroprotection against ischemic challenges is conferred by both ischemic preconditioning (IPC) and preconditioning by activation of the epsilon-isoform of protein kinase C (epsilonPKC-PC). In vivo, ischemic preconditioning enhances GABA release and ameliorates glutamate release during lethal cerebral ischemia. We tested the hypothesis that IPC and epsilonPKC-PC confer neuroprotection by GABA synapses in rat organotypic hippocampal slices. Ischemic preconditioning or epsilonPKC-PC was induced with 15 mins oxygen-glucose deprivation (OGD) or psiepsilonRACK, a selective epsilonPKC activator; and test ischemia consisted of 40 mins OGD. At the time of peak neuroprotection (48 h after preconditioning), we recorded GABA(A) receptor-mediated miniature postsynaptic currents (GABA mPSCs) in vulnerable CA1 pyramidal neurons using whole-cell voltage clamp techniques. The frequency and amplitude of GABA mPSCs significantly increased 48 h after IPC. In contrast, epsilonPKC-PC enhanced only the amplitude of GABA mPSCs with no effect on frequency. We next asked if neuroprotection depended on these changes in GABA synapses. Weak antagonism of the GABA(A) receptor with bicuculline (100 nmol/L) decreased the amplitude of GABA mPSCs by 20.9+/-6.1%. When applied during test ischemia, 100 nmol/L bicuculline abolished neuroprotection conferred by either IPC or epsilonPKC-PC. We conclude that neuroprotection conferred by preconditioning depends on functional modifications of GABA synapses.