1. The nature of the non-adrenergic, non-cholinergic (NANC) transmitter was studied in vitro in the rat duodenum, by use of an isometric-isovolumic preparation. 2. Electrical field stimulation (EFS) induced a tetrodotoxin (TTX)-sensitive fall both in luminal pressure and in isometric tension. 3. Neurotensin (NT) induced TTX-insensitive inhibitory responses similar to those induced by EFS. Vasoactive intestinal peptide (VIP) caused a delayed, slow, concentration-dependent, TTX-insensitive inhibitory effect, detected only by a change in luminal pressure. 4. alpha-chymotrypsin prevented the NT- and VIP-induced inhibitory effects and antagonized the response to EFS. 5. Apamin antagonized the EFS- and NT-induced effects, but failed to affect the relaxation in response to exogenous VIP. 6. Desensitization of NT receptors by exposure to NT (10 nM) for 30 min did not affect the EFS-induced relaxation. 7. These findings provide support for the involvement of a peptide in the NANC relaxation in rat duodenum. However, there is no evidence that NT or VIP are neurotransmitters released from the NANC system in this preparation.