We combined the retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase with choline acetyltransferase immunohistochemistry to study the projections of cholinergic and non-cholinergic neurons of the upper brainstem core to rostral and caudal intralaminar thalamic nuclei, reticular thalamic complex and zona incerta in the cat. After wheat germ agglutinin-horseradish peroxidase injections in the rostral pole of the reticular thalamic nucleus, the distribution and amount of retrogradely labeled brainstem neurons were similar to those found after tracer injection in thalamic relay nuclei (see preceding paper). After wheat germ agglutinin-horseradish peroxidase injections in the caudal intralaminar centrum medianum-parafascicular complex, rostral intralaminar central lateral-paracentral wing, and zona incerta, the numbers of retrogradely labeled brainstem neurons were more than three times higher than those found after injections in thalamic relay nuclei. The larger numbers of horseradish peroxidase-positive brainstem reticular neurons after tracer injections in intralaminar or zona incerta injections results from a more substantial proportion of labeled neurons in the central tegmental field at rostral midbrain (perirubral) levels and in the ventromedial part of the pontine reticular formation, ipsi- and contralaterally to the injection site. Of all retrogradely labeled neurons in the caudal midbrain core at the level of the cholinergic peribrachial area and laterodorsal tegmental nucleus, 45-50% were also choline acetyltransferase-positive after the injections into central lateral-paracentral and reticular nuclei, while only 25% were also choline acetyltransferase-positive after the injection into the centrum medianum-parafascicular complex. These findings are discussed in the light of physiological evidence of brainstem cholinergic mechanisms involved in the blockade of synchronized oscillations and in activation processes of thalamocortical systems.