Effects of ATP on substance P immunoreactivity were examined in cultured dorsal root ganglion neurons. We found that treatment of dorsal root ganglion neurons with ATP significantly depleted substance P immunoreactivity on the neurites and somata of the neurons. The effects of ATP were significantly inhibited by the purinergic P2 receptor antagonists suramin (30 μM) and pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (10 μM). We also showed that ATP-induced depletion of substance P immunoreactivity from dorsal root ganglion neurons depended on the entry of Ca2+. In a spinal cord slice preparation, we also found the internalization of neurokinin-1/substance P receptors in many dorsal horn neurons following the application of ATP or α,β-methylene-ATP. Together these results indicate that activation of P2X receptors may result in release of substance P from primary afferent neurons. © 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.