The objectives of our present experiments were to determine whether the BK(Ca) channel agonist NS1619 is able to induce immediate preconditioning in cultured rat cortical neurons and to elucidate the role of BK(Ca) channels in the initiation of immediate preconditioning. NS1619 depolarized mitochondria and increased reactive oxygen species (ROS) generation, but neither of these effects was inhibited by BK(Ca) channel antagonists. NS1619 also activated the extracellular signal-regulated kinase signaling pathways. One-hour treatment with NS1619 induced immediate protection against glutamate excitotoxicity (viability 24 h after glutamate exposure: control, 58.45+/-0.95%; NS1619 50 microM, 78.99+/-0.90%; NS1619 100 microM, 86.89+/-1.20%; NS1619 150 microM, 93.23+/-1.23%; mean+/-SEM; p<0.05 vs. control; n=16-32). Eliminating ROS during the preconditioning phase effectively blocked the development of cytoprotection. In contrast, the BK(Ca) channel blockers iberiotoxin and paxilline, the phosphoinositide 3-kinase inhibitor wortmannin, the protein kinase C blocker chelerythrine, and the mitogen activated protein kinase antagonist PD98059 were unable to antagonize the immediate neuroprotective effect. Finally, preconditioning with NS1619 reduced the calcium load and ROS surge upon glutamate exposure and increased superoxide dismutase activity. Our results indicate that NS1619 is an effective inducer of immediate neuronal preconditioning, but the neuroprotective effect is independent of the activation of BK(Ca) channels.