There has been controversy over use of selective serotonin reuptake inhibitors (SSRIs) to treat affective disorders in children and adolescents due to clinical reports of increased risk for suicidal ideation and behavior during treatment, and animal studies showing changes in adult anxiety- and depressive-like behaviors after repeated treatment during adolescence. However, the acute effect of serotonergic drugs on affective behavior during adolescence is poorly understood. We investigated serotonergic modulation of anxiety-like behavior in adolescent (PN28-32) and adult (PN67-73) male rats using the SSRI fluoxetine, the 5-HT1A agonist 8-OH DPAT, and the 5-HT2 agonist mCPP. Acute treatment with fluoxetine (10 mg/kg, i.p.) produced greater anxiogenic effects in adults than adolescents in the light/dark (LD) test for anxiety-like behavior, but fluoxetine (2.5, 5, and 10 mg/kg, i.p.) increased extracellular serotonin in the medial prefrontal cortex similarly in both ages. Adults were also more sensitive to the anxiogenic effects of 8-OH DPAT (0.25 and 0.5 mg/kg, i.p.), but not mCPP (0.5 and 1 mg/kg, i.p.), in the LD test. Fluoxetine (10 mg/kg) stimulated greater increases in c-Fos expression across the extended amygdala in adults than in adolescents, and 8-OH DPAT (0.5 mg/kg) produced greater increases in c-Fos in the lateral orbital cortex and central nucleus of the amygdala in adults. These data show that lower anxiogenic effects of acute SSRIs in adolescents are associated with lesser activation of cortical and amygdala brain regions. This immaturity could contribute to the different profile of behavioral effects observed in adolescents and adults treated with SSRIs. © 2013 Elsevier Ltd. All rights reserved.