The retrotrapezoid nucleus (RTN) contains central respiratory chemoreceptors that are inhibited by lung inflation via slowly-adapting pulmonary stretch receptors (SARs). Here we examine whether RTN inhibition by lung inflation could be mediated by a direct projection from SAR second-order neurons (pump cells). Pump cells (N = 56 neurons, 13 rats) were recorded in the nucleus of solitary tract (NTS) of halothane-anesthetized rats with intact vagus nerves. Pump cells had discharges that coincided with lung inflation as monitored by the tracheal pressure. Their activity increased when end-expiratory pressure was raised and stopped instantly when ventilation was interrupted in expiration. Many pump cells could be antidromically activated from RTN (12/36). Nine of those were labeled with biotinamide. Of these 9 cells, eight contained glutamic acid decarboxylase67- (GAD67-) mRNA and seven were found to reside in the lower half of the interstitial subnucleus of NTS (iNTS). Using the retrograde tracer cholera toxin-B, we confirmed that neurons located in or close to iNTS innervate RTN (2 rats). Many such neurons contained GAD67-mRNA and a few contained glycine transporter2 (GLYT2) mRNA. Anterograde tract-tracing with biotinylated dextranamide (4 rats) applied to iNTS also confirmed that this region innervates RTN via a predominantly GABAergic projection. This work confirms that rat many NTS pump cells are located in and around the interstitial subnucleus at area postrema level. We demonstrate that a GABAergic subset of these pump cells innervates the RTN region. We conclude that these inhibitory neurons probably contact RTN chemoreceptors and mediate their inhibition by lung inflation.