Direct actions of nicotine in the CNS appear to be essential for its reinforcing properties. However, activation of nicotinic acetylcholine receptors (nAChRs) on afferent sensory nerve fibers are important components of addiction to, and withdrawal from, cigarette smoking. The present study was to identify the neuroanatomical substrates activated by the peripheral actions of nicotine and to determine whether these sites overlap brain structures stimulated by direct actions of nicotine. Mouse brains were examined by immunohistochemistry for c-Fos protein after intraperitoneal injection of either nicotine (NIC, 30 and 40 µg/kg) and/or nicotine pyrrolidine methiodide (NIC-PM, 20 and 30 µg/kg). NIC-PM induced c-Fos immunoreactivity (IR) at multiple brain sites. In the brainstem, c-Fos IR was detected in locus coeruleus, laterodorsal tegmental nucleus and pedunculotegmental nucleus. In the midbrain, c-Fos IR was observed in areas overlapping the ventral tegmental area (VTA) which includes paranigral nucleus, parainterfascicular nucleus, parabrachial pigmental area and rostral VTA. Other structures of the nicotine brain-reward circuitry activated by NIC-PM included hypothalamus, paraventricular thalamic nucleus, lateral habenular nucleus, hippocampus, amygdala, accumbens nucleus, piriform cortex, angular insular cortex, anterior olfactory nucleus, lateral septal nucleus, bed nucleus of stria terminalis, cingulate and medial prefrontal cortex, olfactory tubercle, medial and lateral orbital cortex. Nicotine, acting through central and peripheral nAChRs, produced c-Fos IR in areas that overlapped NIC-PM induced c-Fos expressing sites. These neuroanatomical data are the first to demonstrate that the CNS structures which are the direct targets of nicotine are also anatomical substrates for the peripheral sensory impact of nicotine.