The medullary respiratory network involves various types of respiratory neurons. The present study focused on possible inhibitory neurons called decrementing expiratory (E-DEC) neurons and aimed to determine whether their transmitter is glycine or GABA. In Nembutal-anesthetized, neuromuscularly blocked, and artificially ventilated rats we labeled E-DEC neurons with Neurobiotin and processed the tissues for detection of mRNA encoding either glycine transporter 2 (GLYT2) as a marker for glycinergic neurons or glutamic acid decarboxylase isoform 67 (GAD67) as a marker for GABAergic neurons, using in situ hybridization. Of 38 E-DEC neurons that were labeled, cranial motoneurons (n = 14), which were labeled as control, were negative for either GLYT2 mRNA (n = 10) or GAD67 mRNA (n = 4). The other E-DEC neurons (n = 24) were non-motoneurons. Sixteen of them were examined for GLYT2 mRNA, and the majority (11 of 16) was GLYT2 mRNA-positive. The remaining E-DEC neurons (n = 8) were examined for GAD67 mRNA, and all of them were GAD67 mRNA-negative. The GLYT2 mRNA-positive E-DEC neurons were located in the ventrolateral medulla spanning the Bötzinger complex (BOT), the rostral ventral respiratory group (VRG), and the caudal VRG. We conclude that not only E-DEC neurons of the BOT but also many E-DEC neurons of the VRG are inhibitory and use glycine as a transmitter. Although the present negative data cannot rule out completely the release of GABA or co-release of glycine and GABA from E-DEC neurons, several lines of evidence suggest that the glycinergic process is primarily responsible for the phasic inhibition of the respiratory network during the expiratory phase.