Using a highly sensitive substance P (SP) radioimmunoassay, we attempted to elucidate the molecular mechanism of SP release from cultured adult rat dorsal root ganglion (DRG) neurons triggered by capsaicin. As a result, we found that capsaicin can induce SP release in the absence of extracellular Ca2+ by activating transient receptor potential vanilloid receptor subtype 1 (TRPV1). Therefore, we compared the pharmacological profile of SP release involved in several intracellular effectors (phosphoinositide 3-kinase (PI3K), Ca2+ release from intracellular stores, and mitogen-activated protein (MAP) kinases) in the presence/absence of extracellular Ca2+ by stimulating DRG neurons with various concentrations (10 to 1,000 nM) of capsaicin. In the presence of extracellular Ca2+, a capsaicin-induced maximal release of SP obtained at 100 nM capsaicin was attenuated by either the phosphoinositide 3-kinase (PI3K) inhibitors (wortmannin and LY294002), the inositol 1,4,5-triphosphate (IP3)-induced Ca2+ release blocker (2-aminoethyl diphenylborinate, 2-APB) or a specific antagonist of TRPV1 (capsazepine). In contrast, in the absence of extracellular Ca2+, only a high concentration (1 microM) of capsaicin induced a significant increase in the SP release, which was then completely abolished by either a mitogen-activated protein kinase kinase (MEK) inhibitor U0126 or capsazepine, and significantly inhibited by either thapsigargin (a Ca2+-ATPase inhibitor) or BAPTA-AM (a rapid Ca2+ chelator). In summary, the activation of TRPV1 by capsaicin modulates the SP release from DRG neurons via two different mechanisms, one requiring extracellular Ca2+, the activation of PI3K and the IP3-dependent intracellular Ca2+ release, and the other which is independent of extracellular Ca2+ but involves the activation of MEK. These data suggest that a distinct SP release mechanism exists on DRG through the activation of TRPV1 without extracellular Ca2+ by capsaicin to facilitate neuropeptide release.