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Following injections of retrograde tracers into the spinal cord enlargements, the mesencephalic locomotor region (MLR) was electrically stimulated to induce locomotion in decerebrate rats. MLR sites coincided with cholinergic pedunculopontine nucleus (PPN) neurons, which were selectively labelled using NADPH diaphorase histochemistry. Retrogradely labelled (spinal-projecting) PPN neurons were scattered among cholinergic PPN neurons. The absence of retrogradely labelled, NADPH diaphorase positive PPN neurons indicates that spinal enlargement projections are non-cholinergic.