The rat retrotrapezoid nucleus (RTN) contains CO₂-activated neurons that contribute to the central chemoreflex and to breathing automaticity. These neurons have two known markers, the transcription factor Phox2b and vesicular glutamate transporter 2 (VGLUT2). Noncatecholaminergic galanin-immunoreactive (ir) neurons within a region of the lower brainstem that seems identical to what is currently defined as the RTN have been previously described. Here we ask whether these galanin-expressing neurons are the same cells as the recently characterized CO₂-sensitive neurons of the RTN. By using in situ hybridization, we found that pre-pro-galanin (PPGal) mRNA is expressed by an isolated cluster of neurons that is co-extensive with the RTN as defined by a population of strongly Phox2b-ir neurons devoid of tyrosine hydroxylase (Phox2b⁺/TH⁻ neurons). This bilateral structure contains about 1,000 PPGal mRNA-positive neurons in the rat. The PPGal mRNA-positive neurons were Phox2b⁺/TH⁻ and as susceptible to destruction by the toxin [Sar⁹, Met (O₂)¹¹]-substance P as the rest of the RTN Phox2b⁺/TH⁻ cells of the RTN. CO₂-activated neurons were recorded in the RTN of anesthetized rats and were labeled with biotinamide. Many of those cells (7/17, 41%, five rats) contained PPGal-mRNA. In conclusion, galanin mRNA is a very specific marker of the glutamatergic Phox2b⁺/TH⁻ neurons of the RTN, but galanin mRNA identifies only half of these putative central respiratory chemoreceptors.