The aims of this study were (1) to characterize calcium signaling in rat cortex induced by repeated in vitro application of the glutamatergic agonists L-glutamate, NMDA, AMPA and DHPG, (2) to analyze the influence of transient severe hypobaric hypoxia (180 Torr) administered in vivo on calcium responses to stimulation of glutamate receptors by their agonists, and (3) to evaluate the effects of preconditioning with intermittent mild hypobaric hypoxia (360 Torr) 24 h before the severe hypoxia, on these Ca2+ responses. Intracellular Ca2+ dynamics was studied using the fluorescent probes fura-2 and chlortetracycline to monitor free and bound calcium (Cai and Cab) respectively. In control cortical slices, application of L-glutamate, NMDA and AMPA induced concomitant increases in Cai and Cab, reflecting Ca2+ influx and its intracellular accumulation in neurons. DHPG, an agonist of group I mGlu receptors induced a decrease in Cab accompanied by a rise in Cai levels, indicating Ca2+ mobilization. In cortical slices collected 24 h after severe hypoxia, the responses of Cab to glutamate administration were increased, DHPG-induced shifts were reversed, the increase in Cab after the first application of AMPA was reduced, while after the second, Cab rises were potentiated, and the increases in Cab evoked by NMDA application were slightly suppressed. The alterations of responses in Cab to the selective agonists were completely prevented by preconditioning with mild hypoxia. Our results suggest that protection of normal glutamatergic calcium signaling contributes to tolerance to hypoxia induced by preconditioning.