The distribution of choline acetyltransferase immunoreactivity in the rat thalamus was investigated by using a specific monoclonal antibody and was compared with the pattern of acetylcholinesterase staining. The only choline acetyltransferase-immunoreactive cell bodies in the thalamus were in the medial habenula. A wide range of densities of immunoreactive fibers and varicosities was found. The highest densities of stained varicosities were in the anteroventral, reticular, lateral mediodorsal, and intralaminar nuclei. At the other extreme, the anterodorsal, ventroposteromedial, and paraventricular nuclei were almost devoid of immunoreactive varicosities. A light density of fibers was observed in several medial nuclei, including parataenial, reuniens, and gelatinosus. Most other nuclei contained moderately dense regions of varicose fibers that were often heterogeneous or patchy. The pattern of choline acetyltransferase immunoreactivity in the thalamus was in general similar to that of acetylcholinesterase. A marked discrepancy, however, was found in the anterodorsal nucleus, which was intensely stained for acetylcholinesterase but contained no apparent choline acetyltransferase immunoreactivity. Numerous physiologic studies have demonstrated striking effects of acetylcholine on thalamic activity. The present study provides a description of choline acetyltransferase-immunoreactive structures in the thalamic nuclei, providing a first step toward elucidating the anatomical basis for the physiologic and functional importance of cholinergic transmission in the thalamus.