The aim of this study was to investigate the degree of functional and anatomical overlap between two major neuronal subpopulations in the rostral ventrolateral medulla: pre-sympathetic (sympathoexcitatory) neurons, and expiratory neurons of the Bötzinger complex. Extracellular recordings were made with dye-filled microelectrodes in pentobarbital anesthetized, paralyzed and artificially ventilated adult Wistar rats. Tests applied included stimulation of baroreceptor afferents, activation of peripheral chemoreceptors and lung stretch receptors, changes in central respiratory drive with hyper- or hypoventilation, nociceptive stimulation, and antidromic stimulation from the T2 segment of the spinal cord or medulla oblongata at obex level. The two groups of neurons showed different patterns of spontaneous activity and generally different responses to these stimuli. The recording positions showed some overlap, but the majority of Bötzinger neurons were dorsolateral to pre-sympathetic neurons. There was a large overlap between the location of pre-sympathetic neurons and the lateral part of the C1 adrenergic group, but only a small overlap between these adrenergic neurons and Bötzinger neurons. These results indicate that the anatomically adjacent pre-sympathetic and Bötzinger expiratory neurons form two functionally distinct neuronal subpopulations.