Abstract: Objective To investigate the effect of hypoxia on the viability of rat brain astrocytes in vitro and determine the optimal duration of hypoxic treatment for studying the biological behavior of the in vitro cultured cells in response to hypoxia. Methods Rat astrocytes were cultured and identified by glial fibrillary acidic protein (GFAP) immunofluorescent staining. After incubation with 5% CO₂+95% N₂ for different time to induce hypoxia, the cells were harvested and observed micros- copically for morphological changes and counting of the dead cells. The culture media of the cells were also collected and pO₂, concentrations of glucose and lactate as well as lactate dehydrogenase (LDH) activity were analyzed. Results As the hypoxia prolonged, the astrocytes appeared swollen and floating in the medium with some becoming necrotic. Compared with the cells in the control group, the changes in the number of necrotic cells, concentrations of glucose and lactate, and LDH activity in the cells of the hypoxic group were significantly different after 10 h of hypoxia (P<0.05). However, the flat and polygonal morphology of the astrocytes almost remained unchanged after 8 h of hypoxia in spite of significant changes in the concentrations of glucose and lactate and LDH activity. Conclusion Hypoxia induced injury and necrosis of rat brain astrocytes in vitro, and 8 h of hypoxia can be the optimal time point for studying the biological behaviors of the cells in response to hypoxia.