Experiments were performed to relate receptor binding to biologic activity for the contractile effect of neurotensin (NT) in guinea pig ileum. The contractile response was examined on pieces of ileum under 1 g tension in a 5 ml bath of oxygenated Tyrode's at 38 degrees C. NT contracted the longitudinal muscle (ED50, approximately 0.3 nM), the 2-3 g response peaking at 1 min and fading rapidly. In the presence of atropine (1 microM), > or = 50% of the response was blocked and the residual effect gave an ED50 of approximately 1.4 nM. In the presence of atropine and CP-96,345, a substance P receptor antagonist (0.2 microM), no contraction was observed at 20 nM NT. Thus, there were two components to the response, one involving acetylcholine (ED50, 0.3 nM) and one substance P (ED50, 1.4 nM). Using membrane preparations and 125I-labeled NT, specific, high affinity receptors for NT were demonstrated in the muscle and myenteric plexus. Scatchard analyses indicated the presence of two binding sites (Kds: approximately 0.1 nM and approximately 2 nM). Sodium ion and GTP analogs inhibited binding. Binding and biologic activity were similar in regard to dependence on specific groups within NT and sensitivity to metal ions. The high potency of Hg++ was consistent with an involvement of free sulfhydryl group(s) in the binding reaction; this was supported by work with SH-directed agents. The results suggest that two receptor types or configurations may mediate the two components of the contractile effect of NT on guinea pig ileum.