Evidence suggests that the vesicular monoamine transporter-2 (VMAT2) is regulated in striatum and dopamine (DA) may play a role in its regulation. DA is an important mediator of the behavioral actions of nicotine, and dopaminergic neurotransmission is altered following nicotine administration. We investigated the effect of nicotine withdrawal on the expression of VMAT2 in the midbrain DA neurons in animals dependent to nicotine. Mice were injected with nicotine free base 2mg/kg, sc, four times daily for 14 days and killed 12-72h after drug discontinuation. VMAT2 protein was increased in the striatum of nicotine-treated mice in a time-dependent fashion at all times studied. Furthermore, in situ hybridization studies demonstrated that VMAT2 mRNA was elevated in the substantia nigra pars compacta and ventral tegmental area, indicating enhanced gene expression and subsequent protein synthesis. Tissue DA content and synthesis were unaltered in the striatum of nicotine-treated mice at the times studied. However, basal DA release was decreased at 12 and 24h after nicotine discontinuation which coincided with the elevated levels of VMAT2 protein. VMAT2 up-regulation might be a compensatory mechanism to restore and maintain synaptic transmission in dopaminergic midbrain neurons during nicotine withdrawal.