Nefazodone HCl (Serzone®) is a new antidepressant with a chemical structure unrelated to selective serotonin reuptake inhibitors (SSRIs), tricyclics, tetracyclics, or monoamine oxidase inhibitors (MAOIs). Nefazodone is active in a number of preclinical tests for antidepressant activity and shows clinical efficacy in the treatment of depression with a more favorable side-effect profile than the structurally similar antidepressant trazodone. Previous studies have shown that nefazodone is a potent antagonist of 5-HT2A receptors and binds to the serotonin transporter in vitro and in vivo. Nefazodone also binds to the norepinephrine transporter in vitro and in acute ex vivo studies. To further investigate the ability of nefazodone to modify serotonergic transmission, the ability of systemically administered nefazodone to inhibit the serotonin transporter was assessed by investigating the ability of nefazodone to prevent p-chloroamphetamine- (PCA) induced depletions of cortical 5-HT concentrations. In addition, the ability of acute and subchronic nefazodone administration to inhibit ex vivo [3H]-5-HT uptake was assessed. Acute administration of nefazodone (30, 100, and 150 mg/kg) antagonized PCA-induced depletion of cortical 5-HT concentrations in a dose-dependent manner at 1,2, and 3 hours post-treatment. This effect was directly correlated with serum nefazodone concentrations. Both 100 mg/kg and 150 mg/kg of nefazodone were equipotent with fluoxetine (10 mg/kg) over the course of the experiment with respect to sparing of 5-HT depletion. Acute administration of nefazodone (100 and 150 mg/kg s.c.) significantly increased the Km, for [3H]-5-HT uptake in rat cortical synaptosomes from 60 nmol/L in controls to 230 and 242 nmol/L in nefazodone-treated rats, respectively. Subchronic administration of nefazodone (100 and 150 mg/kg, s.c., b.i.d. × 5.5 days) reduced [3H]-5-HT uptake by 24% and 29%, respectively. Sub-chronic dosing with fluoxetine (5 mg/kg, s.c., b.i.d. × 5.5 days) reduced [3H]-5-HT uptake by 65% These experiments confirm and extend previous reports concerning the ability of nefazodone to inhibit the 5-HT transporter in vivo.