The Pedunculopontine Nucleus (PPN) is a rostral brainstem structure notable for its population of cholinergic neurones, in addition to glutamatergic and GABAergic cells. In Parkinson’s Disease (PD), the PPN demonstrates degeneration, particularly of the cholinergic population. These losses are thought to underlie deficits in postural stability and gait; two symptoms which have been difficult to treat by conventional PD therapies. Deep Brain Stimulation at the PPN offers promise, but has met with inconsistent results. This may be in part due to targeting inaccuracy, from lack of a detailed anatomical description of the nucleus in humans. Furthermore, recent evidence from rodents finds rostro-caudal clustering of cells in the PPN, thought to reflect functional subterritories. The extent to which these gradients exist in humans (potentially providing new, therapeutically exploitable targets) has been an important, unanswered question. Of equal importance is the determination of PPN anatomy for stereotaxy. Here, a stereological technique was applied to human brainstem tissue (from both controls and PD cases) revealing the presence of hitherto undescribed caudal clustering of cholinergic neurones, with a bimodal (rostral more than caudal) distribution of GABAergic neurones. Glutamatergic cells were found to peak between the poles. PD cases showed a disproportionate loss of cholinergic neurones compared with the other two cell types. The functional impact of cholinergic neuronal loss was studied by behavioural assessment in a lactacystin-lesioned rodent model of PD. Selective chemogenetic stimulation of surviving PPN cholinergic neurones demonstrated significant motor recovery, especially in postural stability and gait. Chemogenetic modulation of the cholinergic PPN was then combined - in a novel approach - with 11C-PHNO bioavailability and Positron Emission Tomography, in the same lactacystin rodent model. This revealed striatal dopamine release follows cholinergic stimulation at the PPN, and implicates the pedunculopontine-nigrostriatal pathway in motor recovery after PPN stimulation.