Inhibitory neurons are important player in the respiratory network of the adult animal, but the functional role of inhibitory neurons for the respiratory rhythm in the developing network is still not understood. Although the complete loss of synaptic inhibition in vivo is lethal, the blockade of glycinergic transmission in slices of the medulla has little effect on the pre-Bötzinger complex network activity. We therefore aimed to investigate whether glycinergic neurons in respiratory medulla can co-release of GABA and glycine. In 20-40% on glycinergic neurons we found mIPSCs have a dual transmitter signature. Furthermore, single cell reverse transcriptase PCR revealed that 46% of glycinergic neurons co-express mRNA of the glycine transporter 2 together with at least one marker protein of GABAergic neurons. Our data suggest that significant co-transmission occurs in the pre-Bötzinger Complex that might be involved in shaping of synaptic inhibition of respiratory glycinergic neurons.