Substance P (SP) is widely distributed in the spinal cord and has been implicated as a neurotransmitter in several spinal cord neuronal systems. To investigate SP receptors in the spinal cord, 125I-Bolton-Hunter-SP (125I-BH-SP) was used to identify and characterize spinal cord binding sites for the peptide. The binding of 125I-BH-SP had the following characteristics: high affinity; time, temperature, and membrane concentration dependent; reversible; and saturable. The IC50 of SP in whole spinal cord was 0.46 nM as compared with 0.95, 60, and 150 nM for physalaemin, eledoisin, and kassinin. Four putative antagonists of SP were less than 0.0001 times as potent as SP in inhibiting 125I-BH-SP binding. IC50s were 5, 7.5, 7.0, and 45 microM for D-Pro2, D-Trp7,9-SP; D-Pro2, D-Phe7, D-Trp9-SP; D-Arg1, D-Pro2, D-Trp7,9, Leu11-SP; and D-Pro4, D-Trp7,9,10-SP(4-11), respectively. The lumbosacral section bound 3 times more SP than the cervical and thoracic sections, although IC50 for the cervical section was 0.06 of that for the lumbosacral and thoracic sections. The data suggest more than one class of binding site for SP in the spinal cord and indicate a direct role for SP in spinal cord functions.