Spontaneous and antagonist-precipitated withdrawal from nicotine is characterized by marked deficits in extracellular dopamine (DA) in striatum, especially in nucleus accumbens, that have been thought to underlie the affective state associated with drug discontinuation. Uptake via the dopamine transporter (DAT) is a key mechanism for regulating the concentrations of extracellular DA. Accordingly, we questioned whether DAT expression and function are altered in striatum in nicotine withdrawal. Male mice, 30-35 g, were injected with nicotine free base 2 mg/kg, s.c., or saline four times daily for 14 days and euthanized 4-72 h after drug discontinuation. DA uptake into striatal synaptosomes was increased 12-24 h into nicotine withdrawal, and accompanied by elevated DAT mRNA in the substantia nigra pars compacta and ventral tegmental area, evaluated by in situ hybridization. The overflow of endogenous DA, measured under basal conditions in striatal slices ex vivo, was decreased during nicotine withdrawal in a time pattern paralleling to that of uptake. Added to striatal slices, the DAT inhibitor nomifensine reduced the observed difference in DA overflow between saline and nicotine withdrawn mice implying a role for the transporter. The presented data suggest that DAT is transiently upregulated in the striatum early during nicotine withdrawal, and enhanced transporter function contributes to the decreased extracellular DA levels.