It is recognized in recent years that activation of delta-opioid receptor (DOR) elicits neuroprotection against hypoxia and ischemia. However, the underlying mechanisms are not well understood yet. Mitochondrial dysfunction plays a key role in hypoxic neuronal injury, but the effect of DOR activation on neurons with a mitochondrial respiratory chain deficiency is poorly elucidated. In this study we tested the effects of DOR activation and inhibition on cultured cortical neurons after inhibiting mitochondrial respiratory chain with sodium azide (NaN(3)) in days 8 cultures. Neuronal injury was assessed by lactate dehydrogenase release. Changes in DOR proteins were investigated using an antibody against the N-terminus of the DOR, which recognizes the 60, 48, and 32 kDa proteins. Our main findings are that 1) delta- but not mu-opioid receptor activation reduces NaN(3)-induced neuronal damage, and this neuroprotective effect is abolished by DOR antagonist (naltrindole, NTI); 2) prolonged DOR inhibition with NTI further increases NaN(3)-induced neuronal damage; 3) NaN(3) treatment down-regulates DOR protein levels in neurons, and the 60 and 32 kDa proteins are particularly sensitive; 4) DADLE, besides activating DOR directly, also reverses the decrease of neuronal DOR protein levels induced by NaN(3), which may contribute greatly to its neuroprotective effect; 5) NTI reverses NaN(3)-induced down-regulation of DOR proteins as well, the effect of NTI amplifying NaN(3)-induced neuronal damage therefore is probably due to its inhibition on DOR activity only. In conclusion, these data suggest that DOR activation plays an important role in neuroprotection against mitochondrial respiratory chain injury.