In this investigation of medullary pathways mediating reflex vagal bradycardia to stimulation of buffer nerves, four series of experiments were done in 37 cats anesthetized with chloralose, paralyzed and artificially ventilated. In the first series using animals in which the contralateral vagus was sectioned and the spinal cord was transected at C2, simultaneous stimulation of the aortic depressor (ADN) and carotid sinus (CSN) nerves elicited a bradycardia of magnitude not significantly different from the algebraic sum of the responses elicited by stimulation of the two buffer nerves separately, suggesting the existence of two separate central pathways mediating vagal bradycardia. In the second series, in spinal unilaterally vagotomized animals, lesions of the nucleus ambiguus (AMB) selectively attenuated the ADN reflex vagal bradycardia but not the CSN response; on the other hand, lesions of the external cuneate nucleus (ECN) attenuated the reflex vagal bradycardia elicited by stimulation of the CSN, but did not alter the ADN response. In the third series of experiments, 153 spontaneously firing single units in the region of the AMB and 98 in the region of the ECN were tested for responses to ADN and CSN stimulation. In the AMB 42% of the responsive units were excited only by stimulation of the ADN, 25% were activated only by stimulation of the CSN and 33% were activated by stimulation of both buffer nerves. Of the single units in the ECN region 85% were excited only by stimulation of the CSN, 15% during stimulation of both the CSN and ADN and none responded to stimulation of only the ADN. In the final series, electrical stimulation of the ECN evoked antidromic compound action potentials in the CSN but not in the ADN. Electrical stimulation of the AMB did not evoke antidromic activity in either the CSN or ADN. These studies provide evidence for the existence of two separate medullary pathways mediating the vagal reflex bradycardia to stimulation of the ADN and CSN.