In this study, we investigated the effects of NMDA receptor antagonists on calcium transients induced by a single 2-min preconditioning anoxia (PA) in rat olfactory cortical slices, and on the ability of PA to prevent pathological calcium overload induced by subsequent 10-min test anoxia (TA). Relative changes in the intracellular Ca2+ concentration (Cai) and in the level of Ca2+ bound to intracellular hydrophobic domains (Cab) were monitored using fura-2 and chlortetracycline, respectively. Our results confirmed that TA induces prominent long-lasting increases in Cai and Cab, reflecting cellular calcium overload. It was found that PA produces moderate increases in both Ca2+ pools and prevents Ca2+ overload induced by TA carried out 90 min later. Calcium transients and the protective effects of PA were significantly suppressed in slices treated with NMDA receptor antagonists during and 30 min after PA. These results indicate that moderate activation of the NMDA receptors participates in the mechanism of the PA-induced anoxic tolerance of cortical neurons.