Microinjections of [3H]choline into the rat centromedian-parafascicular nuclei resulted in retrograde labelling of medium sized neurons in brainstem areas considered to comprise the origin of cholinergic innervation; the ipsilateral pedunculopontine tegmental, laterodorsal tegmental and parabrachial nuclei, and, bilaterally, in bulbar reticular formation. In contrast, D-[3H]aspartate labelled the origin of the corticothalamic input, which is believed to use glutamate and/or aspartate as transmitter. In addition, D-[3H]aspartate labelled smaller cells in mesencephalic and pontine periventricular gray, and parabrachial nuclei. It was concluded that retrograde labelling with [3H]choline and D-[3H]aspartate are selective phenomena with specificity for cholinergic and acidic amino acid connections, although occasional labelling of substantia nigra neurons with [3H]choline indicated that unspecific labelling may occur.