Cells switch to anaerobic glycolysis when there is a lack of oxygen during brain ischemia. Extracellular pH thus drops and such acidosis causes neuronal cell death. The fate of astrocytes, mechanical and functional partners of neurons, in acidosis is less studied. In this in vitro study we investigated the signaling in acidosis-challenged cultured Sprague Dawley neonatal rat cortical astrocytes and whether these signals were related to mitochondrial dysfunction and cell death. Results are presented as means ± S.E.M. and analyzed by ANOVA. Exposure to acidic pH (6.8, 6.0) caused cytosolic Ca2+ elevation (1.3 ± 0.2 and 2.2 ± 0.3 fold of baseline ratio, respectively; fura 2 microfluorimetric assay; 21-34 cells from 4 separate experiments; p < 0.05), p38 MAPK activation and Akt inhibition (Western blot; N = 4). Mitochondrial membrane potential, as measured by JC-1 fluorescence assay, was hyperpolarized after astrocytes were exposed to acidic pH as soon as 1 h and lasted for 24 h (R/G ratio raised to 119 ± 3 % and 148 ± 7 % of control (pH 7.4) at pH 6.8 and 6.0, respectively; N = 3; p < 0.05). Such mitochondrial hyperpolarization was abolished by SC79 (Akt activator; 30 mM; N = 3; p < 0.05) but not by SB203580 (p38 inhibitor; 10 mM) nor by cytosolic Ca2+ chelation by BAPTA (10 mM), suggesting that only the perturbation in Akt signaling was causally related to mitochondrial hyperpolarization. SC79, SB203580 and BAPTA did not alleviate acidic pH-induced cell death (MTT assay; N = 4). Using 2′,7′-dichlorofluorescin diacetate fluorescent assay, pH 6.8 and 6.0 were observed to suppress reactive oxygen species (ROS) production by 31 ± 3 and 53 ± 3 %, respectively; N = 3; p < 0.05), thus ruling out the role of ROS in cytotoxicity. Interestingly, pH 6.8 increased ADP/ATP ratio (ADP/ATP luminescence assay kit) from 0.49 ± 0.03 (pH 7.4 control) to 1.30 ± 0.09 (N = 4; p < 0.05) and caused apoptosis (FITC Annexin V/Propidium iodide detection kit; N = 3); pH 6.0 increased ADP/ATP ratio to 1.79 ± 0.02 (N = 4; p < 0.05) and caused necrosis (FITC Annexin V/Propidium iodide detection kit; N = 3). Therefore, astrocyte cell death in acidosis did not result from mitochondrial potential collapse; in case of acidosis at pH 6.0, necrosis might partly result from mitochondrial hyperpolarization and subsequent suppressed ATP production.