Despite its clinical use for more than two decades, the mechanisms by which trazodone acts as an antidepressant are not clear, because it has affinity for a variety of 5-hydroxytryptamine (5-HT; serotonin) receptors and the 5-HT transporter. This study examined the effects of sustained trazodone administration on 5-HT neurotransmission. Electrophysiological recordings were conducted in anesthetized rats. Subcutaneously implanted minipumps delivered vehicle or trazodone (10 mg/kg/day) for 2 and 14 days. A 2-day trazodone administration suppressed the firing rate of raphe 5-HT neurons, which recovered to baseline after 14 days. This was attributable to 5-HT(1A) autoreceptor desensitization because the suppressant effect of the 5-HT autoreceptor agonist lysergic acid diethylamide was dampened in 14-day trazodone-treated rats. Prolonged trazodone administration did not change the sensitivity of postsynaptic 5-HT(1A) and α(2)-adrenergic receptors in hippocampus, but enhanced synaptic 5-HT levels because the 5-HT(1A) antagonist N-{2-[4 (2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride (WAY-100635) enhanced hippocampal firing in treated rats, but not in controls. Trazodone administration for 14 days increased the 50% recovery time value, an index of 5-HT transporter blockade in vivo, and decreased the inhibitory function of terminal 5-HT(1B) autoreceptors on the electrically evoked release of 5-HT. The agonistic action of trazodone at 5-HT(1A) receptors was characterized as being full because it did not attenuate the inhibitory action of 5-HT when coapplied locally. The enhanced 5-HT neurotransmission by trazodone is caused in part by reuptake blockade and activation of postsynaptic 5-HT(1A) receptors, which may account for its effectiveness in major depression.