Our purpose was to determine if sensitization to nicotine could be assessed using functional magnetic resonance imaging (fMRI) with BOLD contrast. Sensitization describes a phenomenon whereby subsequent doses of a drug produce greater responses than the initial dose. Robust locomotor sensitization was demonstrated in adult male Sprague-Dawley rats by the daily administration of nicotine 0.4 mg/kg over 5 days. In parallel experiments, brain activity was monitored using fMRI in animals receiving their first dose (acute) or fifth dose of nicotine (sensitized) and appropriate saline controls. Compared to the acute nicotine animals, brain activity in the sensitized animals demonstrated prolonged BOLD activation in response to nicotine in the hippocampus, nucleus accumbens, prefrontal cortex, ventral pallidum and ventral tegmentum, and more intense peak activation in the hippocampus, prefrontal cortex and ventral tegmentum. In addition, sensitization was associated with a relative decrease in activation in the anterior cingulate gyrus. Furthermore, despite the rich endowment of nicotinic receptors in the visual cortex there was no change in activation with sensitization, thus establishing the specificity of the observed pattern of regional activation and inhibition. Taken together, the current studies support the premise that nicotine sensitization is accompanied by changes in brain activation including a sensitized BOLD response in the extended limbic system that may subserve the process of dependence.