The 6-hydroxydopamine (6-OHDA) lesion of the rat nigrostriatal pathway is the most widely used animal model of Parkinson’s disease. 6-OHDA is a highly specific neurotoxin which targets catecholamine neurones via the dopamine active transporter (DAT). When injected stereotaxically into the brain, either into the median forebrain bundle (MFB) or into the neostriatum, it causes extensive, irreversible loss of dopamine neurones in the ventral midbrain. The corresponding loss of dopamine innervation in target areas is associated with a range of long-term, behavioural deficits that form the target of experimental therapies, aimed at protecting or restoring dopaminergic deficits. In this chapter, the two most widely used 6-OHDA lesion protocols are described: (1) The MFB lesion that results in >97% unilateral depletion of dopamine neurones, principally in the ipsilateral striatum and nucleus accumbens. (2) The unilateral striatal lesion resulting in partial dopamine denervation of the striatum only. In vivo assessment of both lesion types by drug-induced rotation is also covered.