After our publication in 2014 of the existence of an extrinsic source of acetylcholine to the striatum originated in the brainstem, it raised questions on whether other sources of acetylcholine may have passed unrecognized in the same way. In order to provide a solid and definite answer to these questions, we used conditional tracing in all brain cholinergic cell groups in ChAT::Cre rats and mapped cholinergic axons across the brain. Our results show that brainstem cholinergic axons (pedunculopontine and laterodorsal tegmental nuclei) provide the only extrinsic cholinergic innervation to the dorsal striatum and nucleus accumbens.