Bötzinger (BOTZ) neurons in the rostral ventrolateral medulla fire during the late expiratory phase of the respiratory cycle. These cells inhibit phrenic motor neurons and several types of respiratory neurons in the medulla oblongata. BOTZ cells produce a fast, chloride-mediated inhibition of their target neurons, but the neurotransmitter used by these cells has not been determined. In the present study, we examine whether gamma-aminobutyric acid (GABA) or glycine could be the inhibitory neurotransmitter of BOTZ cells. In chloralose-anesthetized rats, we individually filled 20 physiologically characterized BOTZ neurons with biotinamide by using a juxtacellular labeling method. Medullary sections containing the labeled BOTZ neurons were processed for in situ hybridization by using digoxigenin-labeled riboprobes for glutamic acid decarboxylase isoform 67 (GAD67), a marker for GABAergic neurons, or for glycine transporter 2 (GLYT2), a marker for glycinergic neurons. All BOTZ cells examined contained GLYT2 mRNA (n = 10), whereas none had detectable levels of GAD67 mRNA (n = 10). For a positive control, 12 GABAergic neurons in the substantia nigra pars reticulata also were recorded and filled with biotinamide in vivo. Most of these cells, as expected, had detectable levels of GAD67 mRNA (11 out of 12). These results demonstrate that the juxtacellular labeling method can be combined with in situ hybridization to identify physiologically characterized cells with probable GABAergic or glycinergic phenotypes. Furthermore, these data suggest that BOTZ neurons use the neurotransmitter glycine and not GABA to provide widespread inhibition of respiratory-related neurons.