The enhancement of central serotonin system function underlies the therapeutic effects of selective serotonin reuptake inhibitors (SSRIs), which have become the most commonly used class of antidepressant agents. However, many individuals experience depressive episodes that are resistant to SSRI treatment. Homeostatic mechanisms that limit the extent to which SSRIs enhance serotonergic neurotransmission have been implicated in this phenomenon. Here, we report a novel strategy for enhancing the efficacy of SSRIs. Using in vivo microdialysis methods in rats, the nonselective 5-HT2 receptor antagonist ketanserin was observed to produce a robust augmentation of citalopram-, fluoxetine-, and sertraline-induced elevations of hippocampal extracellular serotonin levels. Similar effects were also observed in cortex. The potentiation of SSRI-induced increases in hippocampal serotonin levels was reproduced by the 5-HT(2C) receptor-selective antagonists SB 242084 and RS 102221, but not by the 5-HT(2A) receptor-selective antagonist MDL 100 907. Although 5-HT(2C) receptor antagonists augmented the actions of SSRIs, they had no effect on extracellular serotonin levels or tail suspension responses when administered alone. These results were in strong accord with independent findings using a line of 5-HT(2C) receptor-null mutant mice. Although this mutation did not affect baseline extracellular serotonin levels or tail suspension test (TST) behavior, it enhanced fluoxetine effects on serotonin levels and immobility in the TST. These findings reveal an unanticipated pharmacological action of 5-HT(2C) receptors that warrants consideration in the development of novel strategies for the treatment of depression.