The retrograde transport technique was utilized in the present study to investigate the afferent projections to the periaqueductal gray of the rat. Iontophoretic injections of horseradish peroxidase were made into the periaqueductal gray of 22 experimental animals and into regions adjacent to the periaqueductal gray in 6 control animal. Utilization of the retrograde transport method permitted a quantitative analysis of the afferent projections not only to the entire periaqueductal gray, but also to each of its four intrinsic subdivisions. The largest cortical input to this midbrain region arises from areas 24 and 32 in the medial prefrontal cortex. The basal forebrain provides a significant input to the periaqueductal gray and this arises predominantly from the ipsilateral lateral and medial preoptic areas and from the horizontal limb of the diagonal band of Broca. The hypothalamus was found to provide the largest descending input to the central gray. Numerous labeled cells occurred in the ventromedial hypothalamic nucleus, the lateral hypothalamic area, the posterior hypothalamic area, the anterior hypothalamic area, the perifornical nucleus and the area of the tuber cinereum. The largest mesencephalic input to the periaqueductal gray arises from the nucleus cuneiformis and the substantia nigra. The periaqueductal gray was found to have numerous intrinsic connections and contained a significant number of labeled cells both above and below the injection site in each case. Other structures containing significant label in the midbrain and isthmus region included the nucleus subcuneiformis, the ventral tegmental area, the locus coeruleus and the parabrachial nuclei. The medullary and pontine reticular reticular formation provide the largest input to the periaqueductal gray from the lower brain stem. The midline raphe magnus and superior central nucleus also supply a significant fiber projection to the central gray. Both the trigeminal complex and the spinal cord provide a minor input to this region of the midbrain. The sources of afferent projections to the periaqueductal gray are extensive and allow this midbrain region to be influenced by motor, sensory and limbic structures. In addition, evidence is provided which indicates that the four subdivisions of the central gray receive differential projections from the brain stem as well as from higher brain structures.