Capsaicin, the pungent component of “hot” chilli peppers, selectively activates a distinct population of primary sensory neurons responsive to noxious stimuli. Many of these fibres express neuropeptides including the tachykinin, substance P. Using cultured dorsal root ganglion neurons, we found that capsaicin (10 μM) stimulated a 2-fold increase in release of substance P in the absence of extracellular Ca²⁺. Elevated potassium (75 mM) was unable to induce release under these conditions. The introduction of Ca²⁺ enhanced capsaicin-induced release and brought about a robust response to potassium. Preincubation of cells with botulinum neurotoxin A (100 nM) completely blocked potassium-induced release but the capsaicin response, in the absence of Ca²⁺, was unaffected. However, toxin treatment dramatically reduced capsaicin-stimulated release in the presence of Ca²⁺. It is concluded that capsaicin induces release of substance P from dorsal root ganglion neurons via two mechanisms, one requiring extracellular Ca²⁺ and the intact synaptosomal-associated protein 25 kDa (SNAP-25), and the other independent of extracellular Ca²⁺ and not involving SNAP-25.