An aqueous extract of brain has recently been shown to contain a factor (substance B) that antagonizes presynaptic inhibition of evoked [3H]acetylcholine release from guinea pig ileal synaptosomes. This factor antagonized the inhibition of electrically evoked contractions of the intact guinea pig longitudinal muscle-myenteric plexus preparation by a variety of pharmacological agents including clonidine, 2-chloroadenosine, morphine and phencyclidine. pA2App values for substance B antagonism of these four agonists were very similar. Schild plot analysis suggests a functional competition between substance B and each of these receptor agonists. In the absence of any agonist, substance B had a minimal effect on the force of contraction. This reversal of inhibition by substance B was not altered by the ganglionic blocking agent hexamethonium. Substance B was not able to reverse the inhibition of contractions elicited by atropine. In addition to its localization in brain, the factor was also found in appreciable quantities in the heart and the ileum, but not in the liver or kidney. These results indicate that an endogenous neuromodulator exists in innervated tissues that antagonizes a subcellular mechanism(s) involved in mediating inhibition of neurotransmitter release by alpha adrenergic, opiate and purinergic agonists, as well as phencyclidine.