We used an antibody to choline acetyltransferase (ChAT) to label cholinergic cells in guinea pig brainstem. ChAT-immunoreactive (IR) cells comprise several prominent groups, including the pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus, and parabigeminal nucleus, as well as the cranial nerve somatic motor and parasympathetic nuclei. Additional concentrations are present in the parabrachial nuclei and superior colliculus. Among auditory nuclei, the majority of ChAT-IR cells are in the superior olive, particularly in and around the lateral superior olive, the ventral nucleus of the trapezoid body and the superior paraolivary nucleus. A discrete group of ChAT-IR cells is located in the sagulum, and additional cells are scattered in the nucleus of the brachium of the inferior colliculus. A group of ChAT-IR cells lies dorsal to the dorsal nucleus of the lateral lemniscus. A few ChAT-IR cells are found in the cochlear nucleus and the ventral nucleus of the lateral lemniscus. The distribution of cholinergic cells in guinea pigs is largely similar to that of other species; differences occur mainly in cell groups that have few ChAT-IR cells. The results provide a basis for further studies to characterize the connections of these cholinergic groups.