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.N euromodulators induce presynaptic signaling to regulate fast neurotransmission triggered by activity-induced Ca 2+ entry into the nerve terminal. Neuromodulators also influence the very low rate of spontaneous quantal release of classical transmitters. However, because spontaneous release is functionally relevant only in specialized cases (1), neuromodulators are not believed to typically induce physiologically significant release in the absence of extracellular Ca 2+ . Studies of endocrine cells suggest that release of peptides packaged in large dense-core vesicles (LDCVs) also requires Ca 2+ entry because of the rarity of spontaneous LDCV fusion and the inefficient permeation of peptides through the LDCV-fusion pore (2, 3). However, because it is difficult to measure peptide release at intact synapses, a much more limited dataset supports the conclusion that Ca 2+ influx into the nerve terminal is absolutely required for peptidergic transmission. Thus, it remains unclear how neuromodulators control synaptic neuropeptide release.With this background in mind, we set out to study regulation of neuropeptide release at the Drosophila neuromuscular junction (NMJ) by octopamine-induced cAMP signaling. Octopamine, the homolog of norepinephrine in Drosophila, controls many behaviors by activating central G protein-coupled receptors that induce adenylyl cyclase activation and intracellular Ca 2+ release (4, 5).