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Botulinum neurotoxin (BoNT) inhibits the release of acetylcholine from presynaptic vesicles through its proteinase activity cleaving the SNARE complex. Parkinson's disease (PD) is associated with locally increased cholinergic activity in the striatum. Therefore, the present study investigates the effect of unilateral intrastriatal BoNT‐A injection in naïve rats on striatal morphology; i.e., the total number of Nissl‐stained neurons and the volume of caudate‐putamen (CPu) were estimated. Furthermore, stainings for markers of gliosis (glial fibrillary acidic protein) and microglia (Iba1) were performed. In addition, the potential beneficial effects of a unilateral intrastriatal injection of BoNT‐A on motor activity in the rat model of hemi‐PD were evaluated. Hemi‐PD was induced by unilateral injection of 6‐hydroxydopamine (6‐OHDA) into the right medial forebrain bundle. Six weeks later, rats received an ipsilateral intrastriatal …