Having previously demonstrated that serotonin (5-HT)-induced chloride secretion in rat distal colon is mediated at both neural and nonneural receptors, we isolated the neural component of this response by adding the selective 5-HT3 receptor agonist, 2-methyl-5-hydroxytryptamine (2Me5HT), to in vitro sheets of rat distal colon with intact neural plexuses. Rats were sacrificed, and the distal colon excised, opened, cut into sections and mounted, all layers intact, as flat sheets in Ussing chambers under short-circuited conditions. 2Me5HT induced a prompt, significant (P < 0.01), concentration-dependent rise in short-circuit current (Isc; EC50 6.2 microM); 50 microM 2Me5HT decreased both net sodium and chloride absorption (-0.1 +/- 0.5 and -2.1 +/- 0.8 muEq/cm2 x hr, respectively); the difference (2.0 +/- 0.8 muEq/cm2 x hr) in these changes was not statistically different from the rise in Isc (1.5 +/- 0.3 muEq/cm2 x hr). Since the only significant change in unidirectional flux was the rise in electrogenic Cl- secretion (P < 0.01), the delta Isc induced by 2Me5HT may be used as a measure of electrogenic chloride secretion induced by the agonist. The rise in Isc induced by 2Me5HT was abolished by both 0.2 microM tetrodotoxin and 0.1 microM ICS 205-930 (a 5-HT3 antagonist) but was not inhibited by 1.0 M atropine 100 microM hexamethonium, 10 microM phentolamine, 10 microM propranolol, 10 microM 5-HTP-DP (a 5-HT1P antagonist), or 0.1 microM ketanserin (a 5-HT2 antagonist). These results indicate that 2-methyl-5-HT is a highly selective agonist for neurally based 5-HT3 receptors in this model.(ABSTRACT TRUNCATED AT 250 WORDS)