Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting ~1% of the population older than 60 years. The administration of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice is one of the most widely used approach to elucidate the mechanisms of cell death involved in PD. Its toxicity is attributed to its active metabolite 1-methyl-4-phenylpyridinium (MPP(+)). However, the magnitude of the PD-like neurodegeneration induced by MPTP depends on many variables, including the route of administration. Different groups, including us, demonstrated that intranasal (i.n.) administration of MPTP constitutes a new route of toxin delivery to the brain that mimics environmental exposure to neurotoxins. In particular, our previous data showed that mice submitted to acute i.n. MPTP administration displayed a significant decrease of striatal dopamine (DA) and a loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. However, little is known about the timing and the anatomical distribution of MPP(+) after i.n. MPTP administration in mice. In the present study, C57BL/6J mice received one dose of i.n. MPTP (1 mg/nostril) and were sacrificed at two different times after the administration. Using matrix-assisted laser desorption-ionization mass spectrometry imaging, a new technique for the detection of endogenous unlabeled molecules in tissue sections, we showed for the first time the MPP(+) anatomical distribution in different brain regions. We demonstrated that the toxin first reached almost all the brain areas; however, in a second time MPP(+) remained highly concentrated in the olfactory bulb, the basal ganglia, the ventral mesencephalon, and the locus coeruleus, regions differently affected in PD.