Abstract Radioligand binding techniques were used to characterize the substance P (SP) binding site on membranes prepared from bovine adrenal medullae. 125I-labelled Bolton-Hunter substance P (BHSP), which recognises the C-terminally directed, SP-preferring NK1 receptor, showed no specific binding. In contrast, binding of [ 3H]SP was saturable (at 6 nM) and reversible, with an equilibrium dissociation constant ( K d) 1.46 ± 0.73 nM, B max 0.73 ± 0.06 pmol/g wet weight and Hill coefficient 0.98 ± 0.01. Specific binding of [ 3H]SP was displaced by SP > neurokinin A (NKA) > SP(3–11) ≈ SP(1–9) > SP(1–7) ≈ SP(1–4) ≈ SP(1–6), with neurokinin B (NKB) and SP(1–3) very weak competitors and SP(5–11), SP(7–11) and SP(9–11) causing negligible inhibition (up to 10 μM). This potency order is quite distinct from that seen with binding to an NK1 site, a conclusion confirmed by the lack of BHSP binding. It appears that Lys 3 and/or Pro 4 are critical for binding, suggesting an anionic binding site. These data suggest the existence of an unusual binding site which may represent a novel SP receptor. This site appears to require the entire sequence of the SP molecule for full recognition.