The origins of the cholinergic and other afferents of several thalamic nuclei were investigated in the rat by using the retrograde transport of wheat germ agglutinin conjugated-horseradish peroxidase in combination with the immunohistochemical localization of choline acetyltransferase immunoreactivity. Small injections placed into the reticular, ventral, laterodorsal, lateroposterior, posterior, mediodorsal, geniculate, and intralaminar nuclei resulted in several distinct patterns of retrograde labelling. As expected, the appropriate specific sensory and motor-related subcortical structures were retrogradely labelled after injections into the principal thalamic nuclei. In addition, other basal forebrain and brainstem structures were also labelled, with their distribution dependent on the site of injection. A large percentage of these latter projections was cholinergic. In the brainstem, the cholinergic pedunculopontine tegmental nucleus was retrogradely labelled after all thalamic injections, suggesting that it provides a widespread innervation to the thalamus. Neurons of the cholinergic laterodorsal tegmental nucleus were retrogradely labelled after injections into the anterior, laterodorsal, central medial, and mediodorsal nuclei, suggesting that it provides a projection to limbic components of the thalamus. Significant basal forebrain labelling occurred only with injections into the reticular and mediodorsal nuclei. Only injections into the reticular nucleus resulted in retrograde labelling of the cholinergic neurons in the nucleus basalis of Meynert. The results provide evidence for an organized system of thalamic afferents arising from cholinergic and noncholinergic structures in the brainstem and basal forebrain. The brainstem structures, especially the cholinergic pedunculopontine tegmental nucleus, appear to project directly to principal thalamic nuclei, thereby providing a possible anatomical substrate for mediating the well-known facilitory effects of brainstem stimulation upon thalamocortical transmission.