The interaction of substance P with human blood T-lymphocytes, which stimulates T-lymphocyte proliferation, was quantified by both flow cytometric and direct binding assays. Fluorescence-detection flow cytometry recorded the binding of dichlorotriazinylamino-fluorescein-labeled substance P to 21 +/- 10% (mean +/- SD, n = 6) and 35 +/- 8% (n = 2) of human blood T-lymphocytes before and after stimulation with 10 micrograms/ml of phytohemagglutinin, respectively. The suppressor-cytotoxic (leu 2a) and helper-inducer (leu 3a) subsets identified by phycoerythrin-labeled monoclonal antibodies contained substance P-reactive T-lymphocytes at respective mean frequencies of 10 and 18%. [3H]substance P bound rapidly and reversibly to a mean of 7035 +/- 2850 sites/T-lymphocyte, which exhibited a dissociation constant (KD) of 1.85 +/- 0.70 X 10(-7) M (mean +/- SD, n = 5). [D-Pro2,D-Phe7,D-Trp9]substance P inhibited the binding of dichlorotriazinylamino-fluorescein-labeled substance P and [3H]substance P to T-lymphocytes at concentrations that suppressed the proliferative response to substance P. Substance P, eledoisin, and substance K (alpha-neurokinin), which all share with substance P the carboxy-terminal substituent -Gly-Leu-Met-NH2, were more potent than substance P in inhibiting the binding of [3H]substance P to T-lymphocytes, suggesting the importance of this sequence in the interaction. Purified human blood B-lymphocytes, monocytes, polymorphonuclear leukocytes, and platelets, and cultured Hut 78 cutaneous lymphoma T-cells, Jurkat cells, Molt-4 lymphoblasts, and HL-60 and U-937 monocyte-like cells all showed only minimal specific binding of [3H]substance P. The recognition of substance P by T-lymphocytes provides one mechanism for selective modulation of immunity by sensory nerves.