Substance P (SP)-immunoreactive (IR) elements were studied in laminae I and II of the chicken spinal cord in conjunction with an anti-SP monoclonal antibody at light- and electron-microscopic levels by means of the indirect antibody peroxidase-antiperoxidase technique. At the light-microscopic level, SP-IR elements were most intensely observed in the dorsolateral portion of the dorsal horn, laminae I and II. Electron-microscopically, SP-IR boutons contained large spherical dense-cored vesicles (diameter range: 60-125 nm) and spherical clear vesicles. They were subdivided into two groups: large SP-IR boutons, which were the central terminals in synaptic glomeruli, and small SP-IR boutons. In the synaptic glomerulus, two kinds of non-IR presynaptic profiles made axo-axonic synapses with the SP-IR central terminal: one was the presynaptic profile containing pleomorphic clear vesicles and the other was the presynaptic profile containing large dense-cored vesicles. A 'septate junction'-like structure was observed between large SP-IR boutons in synaptic glomeruli. The present results suggest that SP-containing primary afferents are modulated presynaptically by two different neurotransmitter or modulator systems.