The presence of substance P in primary afferents that terminate in the outer laminae of the spinal cord has led to considerable interest in the function of this neuropeptide in nociception. We have examined the actions of tachykinin agonists on the membrane potential of neurons in lamina II of a neonatal spinal cord slice preparation in vitro. Only 10.5% (n = 75) of these neurons showed any response to the application of a selective NK1 receptor agonist while 48.3% (n = 60) of neurons in deeper dorsal horn laminae responded to this agonist. Lamina II neurons were equally insensitive to selective NK2 and NK3 agonists. Synaptic potentials evoked in lamina II neurons by peripheral nerve stimulation were similarly not altered by the NK1 agonist. Immunocytochemical studies using an antibody raised against the C-terminal of the NK1 receptor revealed that very few lamina II neurons express NK1 receptors, and this offers an explanation for our findings.