The afferent connections of the mesencephalic reticular formation were studied experimentally in the rat by the aid of the retrograde horseradish peroxidase tracer technique. The results suggest that the rostral portion of the mesencephalic reticular formation receives its main input from the cerebral cortex, the zona incerta and the fields of Forel, the central gray substance, the nuclei reticularis pontis oralis and caudalis, and the deep cerebellar nuclei. Substantial input to the same territory of the mesencephalic reticular formation appears to come from the superior colliculus, the substantia nigra, the parabrachial area, the spinal trigeminal nucleus, and the nucleus reticularis gigantocellularis, whereas several other brain structures, among which the locus coeruleus and the raphe complex, seem to represent modest but consistent additional input sources. The afferentation of more caudal portions of the mesencephalic reticular formation appears to conform to the general pattern outlined above with only three exceptions; the cerebral cortex, the deep cerebellar nuclei and the spinal trigeminal nucleus seem to be relatively modest sources of projections to these levels. Considering that the mesencephalic reticular formation is a critical structure in the "ascending activating systems" the present results, confirming and extending those of many other investigators, characterize a set of pathways that seem to be an important part of the anatomical substrate of the sleep-walking cycle.