Different Parkinson's disease (PD) animal models reproduce the early phase of the disease, which deny the possible existence of a synergic effect of consecutive insults to the dopaminergic neurons. We proposed a novel protocol of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) nigrostriatal lesion, which consists in repeated MPTP intranigral administrations intending to differentiate effects of a single lesion in relation to repeated lesions. For this purpose, a schedule of 3-day intervals between the MPTP administrations, totalizing 3 infusions in 9 days were adopted. A persistent locomotor deficit was produced after the 2nd infusion, remaining until the last time-point. Tyrosine hydroxylase (TH) immunoreactive neurons were reduced in 50% 1 day after the 1st infusion and the neuronal loss remained constant even after the 2nd and 3rd MPTP infusions. In parallel, (TH) protein expression in the substantia nigra pars compacta (SNpc) revealed to be a sensitive target for MPTP, once it was found to be down-regulated immediately after the 1st MPTP exposure until the last time-point. These findings corroborate the concept of an early phase model of PD elicited by MPTP even when this neurotoxin was used according to the protocol currently proposed. The current protocol provided relevant insights about TH expression and irreversible locomotor impairment.