Brain nicotinic receptors display pronounced permeability for Ca2+ and localize to presynaptic nerve terminals, in addition to postsynaptic sites. Chronic exposure to nicotine has been shown to alter brain nicotinic receptor expression, but the functional consequences for presynaptic Ca2+ have not been directly examined. Here, we used confocal imaging to assess Ca2+ responses in individual nerve terminals from cortices of mice treated up to 14 days with nicotine as compared to vehicle-treated controls. Chronic nicotine treatment led to substantially enhanced amplitudes of presynaptic Ca2+ responses to acute application of nicotine at concentrations of 50 nM (2-fold) and 500 nM (1.7-fold), but not 50 μM. In addition, increased expression of high-affinity nicotinic receptors on isolated terminals was observed following chronic treatment, as determined immunocytochemically and pharmacologically. These findings suggest that chronic exposure to nicotine may lead to enhanced sensitivity to nicotine at select presynaptic sites in brain via up-regulation of high-affinity nicotinic receptors.