The innervation sites of the dorsal tegmental acetylcholinesterase (AChE)-containing pathway were examined in rats by combining histochemical and biochemical techniques. A lesion was placed in the nucleus cuneiformis (midbrain reticular formation) and brains were examined after 4 days survival for changes in AChE staining and choline acetyltransferase (ChAT) activity in discrete brain areas. An ipsilateral projection appears to exist to the anterior thalamic nuclei, lateral portion of the medial thalamic nucleus, parafascicular nucleus, pretectal nucleus, posterior thalamic nucleus, and deep layers of the superior colliculus. A possible bilateral innervation to the reticular nucleus of the thalamus and the dorsal and ventral lateral geniculates was found. The parallel use of AChE histochemistry and measurements of ChAT activity in discrete nuclei will be useful for future evaluation of cholinergic pathways.