1. Four putative neurotransmitters (serotonin, substance P, ATP (alpha-beta-methylene-ATP), and vasoactive intestinal peptide, VIP) of the non-adrenergic non-cholinergic (NANC) innervation were examined for their role in the NANC excitatory neurotransmission in channel catfish intestine after adrenergic and cholinergic blockade. 2. VIP at concentrations ranging from 10(-12)M to 10(-4)M did not produce either a relaxant or a contractile response in these segments. 3. Serotonin, substance P and alpha-beta-methyl-ATP produced contractile responses in a dose-dependent manner. Their EC50 values were 5 x 10(-7)M, 5 x 10(-9)M and 5 x 10(-9)M and 5 x 10(-6)M, respectively. 4. Electrical field stimulation of the intestinal segments produced a predominant excitatory response after complete blockade of adrenergic and cholinergic divisions, suggesting a predominant NANC excitatory innervation. 5. Three types of serotonin receptor antagonists, namely methiothepin (predominantly a 5-HT1 antagonist), ketanserin (a selective 5-HT2 antagonist), methysergide and cyproheptadine (predominantly 5-HT2 blockers) and metoclopramide (a selective 5-HT3 blocker) were tested for their effectiveness against serotonin and EFS-induced contractions. Methiothepin, methysergide, cyproheptadine and metoclopramide produced significant blockade of the response to serotonin, whereas only methiothepin and cyproheptadine produced blockade of EFS-induced response. 6. Three agents tested for substance P blockade, namely spantide, 4-11 fragment of substance P, and methysergide (also a serotonin blocker), did not produce significant inhibition of the response to either substance P or EFS. 7. Suramin at a dose that blocked the ED50 concentration of ATP did not produce a significant blockade of the response to EFS suggesting that ATP-involvement in the NANC-e neurotransmission is unlikely. 8. This study confirmed the involvement of serotonin in the expression of non-adrenergic non-cholinergic excitatory response of the channel catfish intestine.