Afferent connections of the anterior thalamic nuclei (ATN) are classically thought to originate in the mammillary body and limbic cortex. This study explores nonlimbic sources of ATN afferents by using retrograde transport of horseradish peroxidase (HRP) to ascertain the relative contribution of these connections. Spread of HRP into adjacent regions was prevented either by removing the overlying cortex or by injecting through permanently implanted cannulas. The main sources of nonlimbic ATN afferents are the pretectum and central gray. Pretectal neurons were HRP-labeled primarily in the contralateral medial pretectal nucleus with a smaller number in the ipsilateral posterior pretectal nucleus. In the central gray, labeled cells were concentrated ipsilaterally in the laterodorsal tegmental nucleus. Additional projections to ATN originate in the reticular and ventral lateral geniculate nuclei of the thalamus, raphe nuclei, peripontine tegmental nucleus, and locus coeruleus. The association of ATN afferents with muscarinic receptors was also explored by means of in vitro receptor autoradiography with the muscarinic ligands propylbenzilylcholine mustard (PrBCM) and pirenzepine (PZ) in normal rats and rats with ablations. Ibotenic acid injections into ATN were used to destroy intrinsic neurons while leaving afferent fibers intact. Whereas such ablations produced statistically significant decreases in PrBCM binding in the anterior dorsal (AD, -45%) and anterior ventral, magnocellular part (AVm, -51%) nuclei, binding in the anterior ventral, parvicellular part (AVp) and anterior medial (AM) nuclei was not significantly decreased. Furthermore, PZ binding in normal rat ATN was significantly less (-72%) than PrBCM binding. These results suggest that a major proportion of muscarinic binding is associated with presynaptic elements. Ibotenic acid ablations of the mammillary body reduced PrBCM binding in ATN whereas lesions in cingulate cortex and laterodorsal tegmental nucleus had no effect. Compared to sham lesion controls, mammillary body lesions resulted in statistically significant decreases in binding bilaterally in AD (-15%), AVm (-19%), and AM (-20%). In conclusion, ATN receive afferents from several nonlimbic regions. Of these inputs, the pretectum may be the primary route through which sensory information reaches ATN. In addition, cholinergic input may modulate activity in projections from the mammillary body to ATN through presynaptic muscarinic receptors.