Synaptic release of neurotransmitters is evoked by activity-dependent Ca 2+ entry into the nerve terminal. However, here it is shown that robust synaptic neuropeptide release from Drosophila motoneurons is evoked in the absence of extracellular Ca 2+ by octopamine, the arthropod homolog to norepinephrine. Genetic and pharmacology experiments demonstrate that this surprising peptidergic transmission requires cAMP-dependent protein kinase, with only a minor contribution of exchange protein activated by cAMP (epac). Octopamine-evoked neuropeptide release also requires endoplasmic reticulum Ca 2+ mobilization by the ryanodine receptor and the inositol trisphosphate receptor. Hence, rather than relying exclusively on activity-dependent Ca 2+ entry into the nerve terminal, a behaviorally important neuromodulator uses synergistic cAMP-dependent protein kinase and endoplasmic reticulum Ca 2+ signaling to induce synaptic neuropeptide release.