Mouse embryonic spinal cord neurons in culture exhibit spontaneous calcium oscillations from day in vitro (DIV) 6 through DIV 10. Such spontaneous activity in developing spinal cord contributes to maturation of synapses and development of pattern-generating circuits. Here we demonstrate that these calcium oscillations are regulated by opioid receptors (KORs). The opioid agonist dynorphin (Dyn)-A (1-13) suppressed calcium oscillations in a concentration-dependent manner, and both the nonselective opioid antagonist naloxone and the -selective blocker norbinaltorphimine eliminated this effect. The KOR-selective agonist (ϩ)--benzeneacetamide (U69593) mimicked the effect of Dyn-A (1-13) on calcium oscillations. A -specific peptide antagonist, zyklophin, was also able to prevent the suppression of calcium oscillations caused by Dyn-A (1-13). These spontaneous calcium oscillations were blocked by 1 M tetrodotoxin, indicating that they are action potential-dependent. Although the L-type voltagegated calcium channel blocker nifedipine did not suppress calcium oscillations, the N-type calcium channel blocker -conotoxin inhibited this spontaneous response. Blockers of ionotropic glutamate receptors, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline and dizocilpine maleate (MK-801), also suppressed calcium oscillations, revealing a dependence on glutamate-mediated signaling. Finally, we have demonstrated expression of KORs in glutamatergic spinal neurons and localization in a presynaptic compartment, consistent with previous reports of KOR-mediated inhibition of glutamate release. The KOR-mediated inhibition of spontaneous calcium oscillations may therefore be a consequence of presynaptic inhibition of glutamate release.