1. In view of its multiple sites of action, we investigated the activity of 5-hydroxytryptamine (5-HT, serotonin) on various potential receptors in the isolated proximal colon of rats. 2. 5-HT induced concentration-dependent contractions of colonic strips (pEC50=7.54+/-0.12). 3. The 5-HT1 receptor agonist, 5-carboxamidotryptamine, induced concentration-dependent contractions (pEC50=5.93+/-0.27); however, neither the 5-HT3 receptor-agonist, phenylbiguanide, nor the 5-HT4 receptor-agonist, renzapride, caused contractions at concentrations as high as 10(-4) M. 4. The 5-HT 1/2 receptor antagonist, methiothepin, caused concentration-dependent nonsurmountable antagonism. The 5-HT3 receptor antagonist, tropisetron, inhibited the contractions to a concentration of 5-HT> or =10(-6) M. Ketanserin had no effect on responses to 5-HT. 5. Tetrodotoxin and atropine had no effect on responses to 5-HT. 6. We conclude that contractions to 5-HT are mediated by 5-HT1-like and probably 5-HT3 receptors that activate tetrodotoxin insensitive mechanisms.