Afferent projections to the periaqueductal gray matter in the rat have been studied by use of the retrograde axonal transport of horseradish peroxidase. Iontophoretic injections of horseradish peroxidase were made in dorsal, lateral and medial areas of the periaqueductal gray, primarily at intercollicular levels. The pattern of projections was similar in all of the injections restricted to the periaqueductal gray. Within the brainstem, numerous reticular formation nuclei were labeled, including nucleus reticularis lateralis, nucleus raphe magnus, pallidus and obscurus, the nucleus reticularis pontis oralis and caudalis, the paralemniscal nucleus and the dorsal and ventral parabrachial nuclei. At diencephalic levels, dense projections were seen from the parafascicular nucleus, dorsal premamillary nucleus, zona incerta, dorsomedial and ventromedial nuclei of the hypothalamus and the retrochiasmatic area, in the ventral portion of the anterior hypothalamus. At forebrain levels, occasional cells were seen in the medial preoptic area, lateral septum and the anterior cingulate cortex. Control injections of horseradish peroxidase into structures adjacent to the periaqueductal gray matter included three well localized deposits in the dorsal raphe. Retrogradely-labeled cells were found in lateral reticular nucleus of the medulla, nucleus raphe magnus, nucleus reticularis pontis caudalis, locus ceruleus, dorsal and ventral parabrachial nuclei, substantia nigra and the lateral hypothalamus. No labeled cells were found in the habenular nuclei. It is suggested that many of the descending hypothalamic and forebrain afferents may be relay centers for descending hippocampal formation efferents. Many of the periaqueductal gray afferent systems receive a direct projection from the hippocampal formation and could therefore coordinate influences from this limbic center with information on homeostatic mechanisms controlled by the hypothalamus. The numerous brainstem afferents to the periaqueductal gray could be involved in relay of ascending sensory information important for initiating any of several behavioral responses known to be controlled by the periaqueductal gray. In addition, certain raphe afferents might play a part in a feedback loop of the pain suppression circuit of which the periaqueductal gray is an important component.