A conjugate (125I-BH-SP) between substance P and 125I-labeled Bolton- Hunter reagent binds reversibly to a single class of high affinity (Kd = 4 nM) binding sites on dispersed rat parotid cells. The total number of binding sites is 49 +/- 17 fmol/mg of protein. The binding affinity of 13 fragments and analogs of substance P correlates with their relative potency in stimulating salivation. The smallest fragment of substance P which exhibits significant binding affinity and saliva- stimulating activity is the COOH-terminal hexa(6–11)peptide. Structurally unrelated neurotransmitters and hormones do not affect the parotid cell binding of 125 I-BH-SP. 125I-BH-SP pre-bound to the cells was recovered mainly in the particulate fractions of the cell homogenate. The 125I-BH-SP binding activity of the parotid cells was inactivated by pretreatment of the cells with papain but not with ribonuclease A or deoxyribonuclease I. Our results suggest that 125I-BH- SP binds to a substance P receptor on parotid membranes and that the receptor, at least in part, may be a protein.