The aim of the present work was to assess the effects of activation of NMDA receptors in rat brain in vivo on calcium homeostasis in isolated non-synaptic brain mitochondria. We have shown recently that acute intoxication with large doses of ammonia leads to activation of NMDA receptors in rat brain in vivo. In the present work we injected rats with ammonium acetate to activate NMDA receptors in vivo and isolated non-synaptic mitochondria to assess calcium homeostasis. We also tested whether blocking NMDA receptors with MK-801 prevents effects on calcium homeostasis induced by ammonium injection. It is shown that activation of NMDA receptors in rat brain in vivo leads to a rapid increase in intramitochondrial calcium content followed by a reduction in the calcium capacity and calcium uptake rate in rat brain mitochondria. Activation of NMDA receptors resulted in increased spontaneous calcium efflux from rat brain mitochondria and in a strong inhibition of Na-induced and tert-butylhydroperoxide-induced calcium efflux. All these effects were prevented by previous blocking of NMDA receptors by injection of MK-801. Cyclosporin A did not affect any of the above parameters, indicating that the mitochondrial permeability transition pore does not play a role in calcium efflux under any of the conditions studied. The results reported indicate that ammonia-induced activation of NMDA receptors in rat brain in vivo alters mitochondrial calcium homeostasis at several different steps.