Enhanced locomotion in a habituated environment is a well documented effect of nicotine mediated by the mesotelencephalic dopaminergic system. The nicotinic receptor subunit alpha6 is, among other subunits, strongly expressed in the dopaminergic neurons of the mesencephalon. To examine the functional role of this subunit, we inhibited its expression in vivo using antisense oligonucleotides. In vitro treatments of embryonic mesencephalic neuron cultures demonstrated that the alpha6 antisense oligonucleotides caused a marked decrease in the level of alpha6 subunit protein. In vivo, 1 week infusion of alpha6 antisense oligonucleotides by osmotic mini-pump reduced the effect of nicotine on locomotor activity in habituated environment by 70%. These data support the notion that the effects of nicotine on the dopaminergic system involve alpha6 subunit containing nAChRs.