The distribution and sources of putative cholinergic fibers within the lateral geniculate nucleus (GL) of the tree shrew have been examined by using the immunocytochemical localization of choline acetyltransferase (ChAT). ChAT-immunoreactive fibers are found throughout the thalamus but are particularly abundant in the GL as compared to other principal sensory thalamic nuclei (medial geniculate nucleus, ventral posterior nucleus). Individual ChAT-immunoreactive fibers are extremely fine in caliber and display numerous small swellings along their lengths. Within the GL, ChAT-immunoreactive fibers are more numerous in the layers than in the interlaminar zones and, in most cases, the greatest density is found in layers 4 and 5. Two sources for the ChAT-immunoreactive fibers in the GL have been identified--the parabigeminal nucleus (Pbg) and the pedunculopontine tegmental nucleus (PPT)--and the contribution that each makes to the distribution of ChAT-immunoreactive fibers in GL was determined by combining immunocytochemical, axonal transport, and lesion methods. The projection from the Pbg is strictly contralateral, travels via the optic tract, and terminates in layers 1, 3, 5, and 6 as well as the interlaminar zones on either side of layer 5. The projection from PPT is bilateral (ipsilateral dominant) and terminates throughout the GL as well as in other thalamic nuclei. Lesions of the Pbg eliminate the ChAT-immunoreactive fibers normally found in the optic tract but have no obvious effect on the density of ChAT-immunoreactive fibers in the contralateral GL. In contrast, lesions of PPT produce a conspicuous decrease in the number of ChAT-immunoreactive fibers in the GL and in other thalamic nuclei on the side of the lesion but have no obvious effect on the number of ChAT-immunoreactive fibers in the optic tract. These results suggest that there are two sources of cholinergic projections to the GL in the tree shrew which are likely to play different roles in modulating the transmission of visual activity to the cortex. The Pbg is recognized as a part of the visual system by virtue of its reciprocal connections with the superficial layers of the superior colliculus, while the PPT is a part of the midbrain reticular formation and is thought to play a non-modality-specific role in modulating the activity of neurons throughout the thalamus and in other regions of the brainstem.