5-Hydroxytryptamine (5-HT) caused a persistent, concentration-dependent increase of spontaneous release of endogenous dopamine (DA) from superfused rat striatal slices. 2-Methyl-5-HT, a selective 5-HT3 agonist, mimicked the 5-HT response with a potency only slightly less than that of 5-HT. A highly selective 5-HT3 antagonist, ICS 205-930 [(3-alpha-tropanyl)1H-indole-3-carboxylic acid ester], inhibited the effect of both agonists with a pKB value characteristic of 5-HT3 receptors. 5-HT-evoked DA release was resistant to antagonism by methiothepin and methysergide, antagonists at 5-HT 1-like and 5-HT2 receptors. Neither (2,5-dimethoxy-4-iodophenyl)-2-aminopropane, the selective 5-HT2 receptor agonist, nor 5-carboxamidotryptamine, the selective 5-HT 1-like receptor agonist, altered DA release. The release of DA by 5-HT3 stimulation was Ca++-dependent and partially sensitive to tetrodotoxin. 5-HT and 2-methyl-5-HT also increased K+-evoked DA release. These observations constitute direct, unambiguous evidence that in rat striatum 5-HT3 receptors modulate release of DA.