The efferent projections of the periaqueductal gray in the rabbit have been described by anterograde tract-tracing techniques following deposits of tritiated leucine, or horseradish peroxidase, into circumscribed sites within dorsal, lateral or ventral periaqueductal gray. No attempts were made to place labels in the fourth, extremely narrow (medial), region immediately surrounding the aqueduct whose size and disposition did not lend itself to confined placements of label within it. These anatomically distinct regions, defined in Nissl-stained sections, corresponded to the same regions into which deposits of horseradish peroxidase were made in order for us to describe afferent projections to the periaqueductal gray. In this present study distinct ascending and descending fibre projections were found throughout the brain. Terminal labelling was detected in more than 80 sites, depending somewhat upon which of the three regions of the periaqueductal gray received the deposit. Therefore, differential projections with respect to both afferent and efferent connections of these three regions of the periaqueductal gray have now been established. Ventral deposits disclosed a more impressive system of ramifying, efferent fibres than did dorsal or lateral placements of labels. With ventral deposits, ascending fibres were found to follow two major pathways from periaqueductal gray. The periventricular bundle bifurcates at the level of the posterior commissure to form hypothalamic and thalamic components which distribute to the anterior pretectal region, lateral habenulae, and nuclei of the posterior commissure, the majority of the intralaminar and midline thalamic nuclei, and to almost all of the hypothalamus. The other major ascending pathway from the periaqueductal gray takes a ventrolateral course from the deposit site through the reticular formation or, alternatively, through the deep and middle layers of the superior colliculus, to accumulate just medial to the medial geniculate body. This contingent of fibres travels more rostrally above the cerebral peduncle, distributing terminals to the substantia nigra, ventral tegmental area and parabigeminal nucleus before fanning out and turning rostrally to contribute terminals to ventral thalamus, subthalamus and zona incerta, then continuing on to supply amygdala, substantia innominata, lateral preoptic nucleus, the diagonal band of Broca and the lateral septal nucleus. Caudally directed fibres were also observed to follow two major routes. They either leave the periaqueductal gray dorsally and pass through the gray matter in the floor of the fourth ventricle towards the abducens nucleus and ventral medulla, or are directed ventrally after passing through either the inferior colliculus or parabrachial nucleus. These ventrally directed fibres merge just dorsal to the pons on the ventral surface of the brain.(ABSTRACT TRUNCATED AT 400 WORDS)