We tested whether the protection of hypoxic neurons by the inhaled anesthetic isoflurane is related to the Ca2+-dependent phosphorylation of MAP kinases and anti-apoptotic co-factors. In cultures of mouse cortical neurons we measured changes in the phosphorylation of Ca2+-dependent and Ca2+-independent MAP kinases, transcription factors, and apoptosis regulators after hypoxia or hypoxia combined with isoflurane (1% in gas phase). In hypoxic neurons, isoflurane reduced cell death and TUNEL staining by >80%. Isoflurane released Ca2+ from intracellular stores, increasing [Ca2+]i in oxygenated neurons by approximately 20%. Neuroprotection was associated with a smaller increase in [Ca2+]i in hypoxic neurons and required IP3 receptors and phospholipase C. In hypoxic neurons, isoflurane increased the phosphorylation of the Ca2+-dependent MAP kinases Pyk2 and p42/44 (ERK). The Ca2+-independent MAP kinase p38 pathway showed increased phosphorylation with isoflurane but not with ionomycin, a Ca2+ ionophore. JNK was phosphorylated in hypoxic neurons in the presence of isoflurane, as was the transcription factor c-Jun; JNK inhibition with SP600125 prevented both phosphorylation of c-Jun and neuroprotection. Isoflurane decreased phosphorylation of the pro-apoptotic cofactors Bad and p90RSK and increased Akt phosphorylation. However, with the exception of c-Jun, transcription factors (Elk-1, GSK-3, Forkhead, p90RSK) decreased or remained unchanged. We conclude that isoflurane's protection of hypoxic cortical neurons involves signaling that includes changes in intracellular Ca2+ regulation, several MAP kinase pathways and modulation of apoptosis regulators.