The distribution of neurons, fibers and terminal fields in rat brainstem displaying positive immunoreactivity to a polyclonal antiserum to human placental choline acetyltransferase (ChAT) is described. The antiserum was used at the high dilution of 1:10,000 and was coupled with a sensitive detection system using the nickel ammonium sulfate intensification method. In addition to previously described ChAT immunopositive groups of large cells in the cranial motor nuclei, and the parabrachial and reticular complexes, many small or medium size, weakly immunopositive neurons were identified. Some of these appeared in structures in the region of the fourth ventricle, including the area postrema. Others were in structures associated with the superior olivary complex, including the lateral superior olive, and the medioventral, lateroventral and superior periolivary nuclei. Scattered, weakly positive cells were seen in numerous other structures, including the ventral tegmental area of Tsai, central gray, superior colliculus, spinal nucleus of nerve 5, dorsal cochlear nucleus and non-motor regions of the spinal cord. The prominent ascending fiber tract of the laterodorsal tegmental pathway was traceable from the parabrachial area to the subgeniculate region of the thalamus. Prominent terminal fields were seen in a number of brainstem structures, including the superior colliculus, pontine nuclei, anterior pretectal nucleus, interpeduncular nucleus and spinal nucleus of nerve 5. The association of small ChAT positive cells and terminal fields with many sensory structures suggests a significant cholinergic participation in the physiology of sensory function.