The role of serotonin in the effect of amphetamine on the firing rate of midbrain dopaminergic neurons was examined using unit recordings of identified A10 dopamine neurons in the chloral hydrate-anesthetized rat. Amphetamine (1 mg/kg, i.v.) reduced the firing rate of these neurons approximately 50 to 60%. This effect was blocked in animals pretreated with the selective serotonin-2 (5-HT2) receptor antagonists, MDL 28,133A (0.2 mg/kg, i.v.) or ritanserin (1 mg/kg, i.v.). Although pretreatment with L-dopa (100 mg/kg, i.v.) plus carbidopa (25 mg/kg, i.p.) alone had no effect on amphetamine-induced slowing of A10 dopamine neurons, when coadministered with the 5-HT2 antagonists, the dopamine precursor completely restored this amphetamine-induced slowing. To verify the role of serotonin in these findings, rats were pretreated with the tryptophan hydroxylase inhibitor, p-chlorophenylalanine (250 mg/kg/day for 2 days) to deplete cortical serotonin levels. Consistent with the results observed with the 5-HT2 receptor antagonists, amphetamine did not produce a significant reduction in the firing rate of A10 neurons in serotonin-depleted rats. These results suggest that, under some conditions, serotonergic input via the activation of 5-HT2 receptors may regulate the availability of the pool of dopamine, which is subject to amphetamine release.