Transient exposure of rat cortical cultures to nonlethal oxygen-glucose deprivation (OGD-preconditioning) induces tolerance to otherwise lethal oxygen-glucose deprivation (OGD) or N-methyl-D-aspartate 24 h later. This study evaluates the role of cytosolic and mitochondrial Ca²⁺-dependent cellular signaling. Mechanistic findings are placed in context with other models of ischemic preconditioning or known neurotoxic pathways within cortical neurons. Tolerance to otherwise lethal OGD is suppressed by performing OGD-preconditioning in the presence of the broad-scope catalytic antioxidants Mn(III)tetra(4-carboxyphenyl)porphyrin (MnTBAP) or Zn(II) tetra(4-carboxyphenyl) porphyrin (Zn(II)TBAP), but not by a less active analogue, Mn(III)-tetra(4-sulfonatophenyl)porphyrin, or a potent superoxide scavenger, Mn(III)-tetra(N-ethyl-2-pyridyl)porphyrin chloride. Inhibitors of adenosine A1 receptors, nitric oxide synthase, mitogen-activated protein kinase and poly(ADP-ribose) polymerase fail to suppress OGD-preconditioning, despite possible links with reactive oxygen species in other models of ischemic preconditioning. Preconditioning is suppressed by diisothiocyanostilbene-2,2'-disulphonate (DIDS), which has been ascribed elsewhere to inhibition of superoxide transport to the cytosol through mitochondrial anion channels. However, although inducing mitochondrial Ca²⁺ uptake, neuronal preconditioning is largely insensitive to mitochondrial uncoupling with carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone or 2,4-dinitrophenol. Uncouplers will prevent production of mitochondrial reactive oxygen species, implying non-mitochondrial targets by MnTBAP, Zn(II)TBAP and DIDS. Emphasizing the importance of an increase in cytosolic Ca²⁺ during preconditioning, a Ca²⁺/calmodulin-dependent protein kinase II inhibitor, KN-62, suppresses development of subsequent tolerance. Summarizing, only those cellular transduction pathways which have the potential to be neurotoxic may be activated by preconditioning in cortical neurons. Finally, a marked decrease in extracellular [glutamate] is observed during otherwise lethal OGD in preconditioned cultures, suggesting that this end effector may represent a point of convergence across different preconditioning models.