Activation of opioid receptors in the CNS evokes a dramatic decrease in heart rate which is mediated by increases in inhibitory parasympathetic activity to the heart. Injection of opiates into the nucleus ambiguus, where premotor cardiac parasympathetic nucleus ambiguus neurons are located elicits an increase in parasympathetic cardiac activity and bradycardia. However, the mechanisms responsible for altering the activity of premotor cardiac parasympathetic nucleus ambiguus neurons is unknown. This study examined at the electron microscopic level whether premotor cardiac parasympathetic nucleus ambiguus neurons possess postsynaptic opioid receptors and whether mu-opioid receptor agonists alter voltage-gated calcium currents in these neurons. Premotor cardiac parasympathetic nucleus ambiguus neurons were identified in the rat using retrograde fluorescent tracers. One series of experiments utilized dual-labeling immunocytochemical methods combined with electron microscopic analysis to determine if premotor cardiac parasympathetic nucleus ambiguus neurons contain mu-opioid receptors. In a second series of experiments whole cell patch clamp methodologies were used to determine whether activation of postsynaptic opioid receptors altered voltage-gated calcium currents in premotor cardiac parasympathetic nucleus ambiguus neurons in brainstem slices. The perikarya and 78% of the dendrites of premotor cardiac parasympathetic nucleus ambiguus neurons contain mu-opioid receptors. Voltage-gated calcium currents in premotor cardiac parasympathetic nucleus ambiguus neurons were comprised nearly entirely of omega-agatoxin-sensitive P/Q-type voltage-gated calcium currents. Activation of mu-opioid receptors inhibited these voltage-gated calcium currents and this inhibition was blocked by pretreatment with pertusis toxin. The mu-opioid receptor agonist endomorphin-1, but not the mu-opioid receptor agonist endomorphin-2, inhibited the calcium currents. In summary, mu-opioid receptors are located postsynaptically on premotor cardiac parasympathetic nucleus ambiguus neurons. The mu-opioid receptor agonist endomorphin1 inhibited the omega-agatoxin-sensitive P/Q-type voltage-gated calcium currents in premotor cardiac vagal nucleus ambiguus neurons. This inhibition is mediated via a G-protein mediated pathway which was blocked by pretreatment with pertusis toxin. It is possible that the inhibition of calcium currents may act to indirectly facilitate the activity of premotor cardiac parasympathetic nucleus ambiguus neurons by disinhibition, such as by a reduction in inhibitory calcium activated potassium currents.