The marginal layer (ML) that lines the ventral surface of the medulla oblongata (VMS) contains neurons thought to contribute to central chemoreception, the process by which systemic hypercapnia activates respiration. The transmitters and connectivity of ML neurons are poorly known. The present study focuses on a group of nonserotonergic ML neurons, often located in close proximity to the entry point of penetrating blood vessels. These neurons (approximately 300/brain) contain vesicular glutamate transporter2 (VGLUT2) mRNA and are thus probably glutamatergic. They cluster below the caudal half of the facial motor nucleus, lateral to the serotonergic cells of the ML. The projections of serotonergic and nonserotonergic ML neurons were investigated by retrograde labeling with Fluoro-Gold. ML VGLUT2 mRNA-expressing neurons lack spinal projections and innervate the dorsolateral pons and the ipsilateral ventral respiratory column (VRC), most particularly, the region of the pre-Bötzinger complex and rVRG. The latter two regions receive a very small input from ML serotonergic neurons which, instead, heavily innervate the spinal cord. In conclusion, a small region of the VMS marginal layer contains glutamatergic neurons that innervate the main respiratory centers of the medulla oblongata and pons. These glutamatergic neurons are located in a chemosensitive region of the ML and their projections are consistent with a role in central chemoreception. The serotonergic neurons of the ML, though known to be activated by CO(2), probably do not contribute to central chemoreception, given that they innervate sympathetic efferents and project at best very lightly to the VRC.