Organization of locomotor behavior is altered in mice knockout for the β2 subunit of the nicotinic receptor—β2−/− mice—during novelty exploration. We investigated the neuronal basis of this alteration by measuring activation of the immediate early gene c-fos in the brains of wild-type (WT) and β2−/− mice after exploration of a novel or a familiar environment. Results show 1) no constitutive difference between WT and β2−/− mice in c-fos gene expression in any brain region, 2) novelty exploration triggered activation of the hippocampus and the reward circuit while exploration of a familiar environment produced increased activation in the amygdala, and 3) in β2−/− mice, exploration of novelty, but not familiarity, induced an increase in activation in the prelimbic prefrontal cortex (PFC) compared with WT mice. c-Fos immunoreactivity after different stages of learning in a maze increased similarly in the prelimbic area of both WT and β2−/− mice, while their performance differed. In WT mice, exploration of a novel environment triggered an increase in c-Fos expression in the reward circuit and the hippocampus, while in β2−/− mice, the amygdala and the motor cortex were additionally activated. We also highlight the role of nicotinic receptors during activation of the PFC, specifically during free exploration of a novel environment.