The ventrolateral medulla oblongata (VLM) of the brainstem contains neurochemically heterogeneous neurons that have a critical role in cardiovascular and respiratory regulation. Previous anatomical studies have shown the existence of galanin immunoreactivity in the medulla oblongata, but a detailed characterization is lacking. In this study, we demonstrate three populations of preprogalanin mRNA (PPG)-expressing neurons in the VLM of the adult, male Sprague-Dawley rat: a retrotrapezoid nucleus (RTN) group, a group in the rostral ventral respiratory group (rVRG), and a subpopulation of A1 neurons. PPG+ neurons express tyrosine hydroxylase (TH) only in the A1 region of the VLM, where approximately 56% of PPG+ neurons contain TH (79 ± 14; n = 4). PPG+ neurons do not express vesicular acetylcholine transporter (vAChT) in the VLM (n = 3). However, 33% of PPG+ neurons contain neurokinin-1 receptor (NK1R) in the rVRG (126 ± 12; n = 12), accounting for ∼28% of all NK1R+ neurons in the region. Retrogradely transported cholera toxin B injected into the thoracic spinal cord (T1) revealed that bulbospinal PPG+ neurons are present in the rVRG (n = 3; ∼26% of PPG+ neurons). PPG+ neurons in the RTN and locus coeruleus are selectively activated (Fos) following 2 hours of exposure to hypercapnia, but not by hypoxia. Neurons in the A1, nucleus of the solitary tract, and dorsomedial hypothalamus are activated by both chemoreceptor stimuli. The results suggest that PPG+ neurons represent a population of brainstem neurons that play a critical and differential role in the chemoreflex responses to hypoxia and hypercapnia. J. Comp. Neurol., 2012. © 2011 Wiley Periodicals, Inc.