We investigated the organization of projections from the rat midbrain periaqueductal gray to nucleus ambiguus and the periambigual region using retrograde and anterograde tract tracing techniques. Retrograde tracing results revealed that neurons that project to nucleus ambiguus arise from three discrete, longitudinally organized columns of neurons located in the supraoculomotor central gray, lateral and ventrolateral periaqueductal gray. Anterograde tracing studies demonstrated that projections from these three columns of periaqueductal gray neurons terminate with topographic specificity in nucleus ambiguus and the periambigual region. Double-labelling studies demonstrated that periaqueductal gray neurons terminate in close contiguity to cholinergic neurons in the compact, semicompact, loose and external formations of nucleus ambiguus. The present results suggest that projections from periaqueductal gray to nucleus ambiguus may mediate, in part, certain cardiovascular adjustments and vocalizations produced by stimulation of periaqueductal gray.