alpha-Latrotoxin, from black widow spider venom, stimulates exocytosis of small synaptic vesicles at central and peripheral synapses. However, it is widely accepted that neuropeptide-containing large dense-core vesicles are insensitive to the toxin. In the present study, we investigated whether alpha-latrotoxin releases neuropeptides from primary afferent and enteric neurons. The guinea-pig renal pelvis is innervated by primary sensory neurons containing tachykinins and calcitonin gene-related peptide, but has no functional cholinergic or noradrenergic motor innervation. alpha-Latrotoxin increased the amplitude of spontaneous myogenic contractions of the renal pelvis, and this effect was prevented by prior capsaicin desensitization and by antagonists at neurokinin-1 and neurokinin-2 receptors. In the presence of the latter antagonists, alpha-latrotoxin decreased the amplitude of the contractions, and this is likely to be mediated by calcitonin gene-related peptide. Thus, alpha-latrotoxin releases tachykinins and calcitonin gene-related peptide from capsaicin-sensitive sensory neurons in the renal pelvis. The circular muscle of the guinea-pig distal colon is innervated by excitatory and inhibitory motor neurons, which use a number of transmitters. In the presence of antagonists to block each of the known transmitters apart from the tachykinins, alpha-latrotoxin increased the amplitude of spontaneous contractions; this effect was prevented by the prior addition of neurokinin-1 and neurokinin-2 receptor antagonists. Thus, alpha-latrotoxin stimulates the release of tachykinins from excitatory motor neurons in the myenteric plexus of the distal colon. In conclusion, this study demonstrates that alpha-latrotoxin is able to evoke the release of neuropeptides from both sensory and enteric neurons. This suggests that exocytosis of large dense-core vesicles shares more of the features of exocytosis of small synaptic vesicles than has previously been appreciated.