The protective potential of nortriptyline has been reported in a few experimental models of brain ischemia, both in vivo and in vitro. However, the detailed molecular mechanisms of the protective action of the drug are still unresolved. The aim of the present study was to determine whether treatment with low or medium concentrations of nortriptyline (0.1-10 μM) might have an effect on cPLA₂ protein and/or mRNA expression in ischemic astrocytes, and whether this influence might be related to its potential positive influence on cell viability. On the 21(st) day in vitro, primary cultures of rat cortical astrocytes were subjected to ischemia-simulating conditions (combined oxygen glucose deprivation, OGD) for 24 h and exposed to nortriptyline. The drug at concentrations of 0.1 and 1 μM attenuated the expression of cPLA₂ (both the phosphorylated and unphosphorylated forms) together with a significant decrease in the cPLA₂ mRNA level in ischemic astrocytes. We have demonstrated that nortriptyline influences a decrease in cPLA₂-mediated arachidonic acid (AA) release through a mechanism that appears to involve the attenuation of both PKC and Erk1/2 kinase expression. Nortriptyline also significantly prevented mitochondrial depolarization in ischemic astrocytes. Moreover, the antidepressant protected glial cells against OGD-induced apoptosis and necrosis. Our findings document a role for cPLA₂ expression attenuation and AA release inhibition in the protective effect of nortriptyline in ischemic astrocytes.