The vesicular monoamine transporter type 2 gene (VMAT2) has a crucial role in the storage and synaptic release of all monoamines, including serotonin (5-HT). To evaluate the specific role of VMAT2 in 5-HT neurons, we produced a conditional ablation of VMAT2 under control of the serotonin transporter (slc6a4) promoter. VMAT2(sert-cre) mice showed a major (-95%) depletion of 5-HT levels in the brain with no major alterations in other monoamines. Raphe neurons contained no 5-HT immunoreactivity in VMAT2(sert-cre) mice but developed normal innervations, as assessed by both tryptophan hydroxylase 2 and 5-HT transporter labeling. Increased 5-HT(1A) autoreceptor coupling to G protein, as assessed with agonist-stimulated [(35)S]GTP-γ-S binding, was observed in the raphe area, indicating an adaptive change to reduced 5-HT transmission. Behavioral evaluation in adult VMAT2(sert-cre) mice showed an increase in escape-like reactions in response to tail suspension and anxiolytic-like response in the novelty-suppressed feeding test. In an aversive ultrasound-induced defense paradigm, VMAT2(sert-cre) mice displayed a major increase in escape-like behaviors. Wild-type-like defense phenotype could be rescued by replenishing intracellular 5-HT stores with chronic pargyline (a monoamine oxidase inhibitor) treatment. Pargyline also allowed some form of 5-HT release, although in reduced amounts, in synaptosomes from VMAT2(sert-cre) mouse brain. These findings are coherent with the notion that 5-HT has an important role in anxiety, and provide new insights into the role of endogenous 5-HT in defense behaviors.