Previous anatomical and physiological studies have revealed a substantial projection from the periaqueductal gray (PAG) to the nucleus paragigantocellularis (PGi). In addition, physiological studies have indicated that the PAG is composed of functionally distinct subregions. However, projections from PAG subregions to PGi have not been comprehensively examined. In the present study, we sought to examine possible topographic specificity for projections from subregions of the PAG to PGi. Pressure or iontophoretic injections of wheat germ agglutinin-conjugated horseradish peroxidase, or of Fluoro-Gold, placed into the PGi of the rat retrogradely labeled a substantial number of neurons in the PAG from the level of the Edinger-Westphal nucleus to the caudal midbrain. Retrogradely labeled neurons were preferentially aggregated in distinct subregions of the PAG. Rostrally, at the level of the oculomotor nucleus, labeled neurons were i) compactly aggregated in the ventromedial portion of the PAG corresponding closely to the supraoculomotor nucleus of the central gray, ii) in the lateral and ventrolateral PAG, and iii) in medial dorsal PAG. More caudally, retrogradely labeled neurons became less numerous in the dorsomedial PAG but were more widely scattered throughout the lateral and ventrolateral parts of the PAG. Only few retrogradely labeled neurons were found in the ventromedial part of the PAG at caudal levels. Injections of retrograde tracers restricted to subregions of the PGi suggested topography for afferents from the PAG. Injections into the lateral portion of the PGi yielded the greatest number of labeled neurons within the rostral ventromedial PAG. Medially placed injections yielded numerous retrogradely labeled neurons in the lateral and ventrolateral PAG. Injections placed in the rostral pole of the PGi (medial to the facial nucleus) produced the greatest number of retrogradely labeled neurons in the dorsal PAG. To examine the pathways taken by fibers projecting from PAG neurons to the medulla, and to further specify the topography for the terminations of these afferents in the PGi, the anterograde tracer Phaseolus vulgaris-leucoagglutinin was iontophoretically deposited into subregions of the PAG that contained retrogradely labeled neurons in the above experiments. These results revealed distinct fiber pathways to the rostral medulla that arise from the dorsal, lateral/ventrolateral, and ventromedial parts of the PAG. These injections also showed that there are differential but overlapping innervation patterns within the PGi. Consistent with the retrograde tracing results, injections into the rostral ventromedial PAG near the supraoculomotor nucleus yielded anterograde labeling immediately ventral to the nucleus ambiguus in the ventrolateral medulla, within the retrofacial portion of the PGi.(ABSTRACT TRUNCATED AT 400 WORDS)