The respiratory rhythm is generated and regulated by a neuronal network within the lower brainstem. While the neuronal mechanisms of rhythm generation have been extensively investigated, the contribution of glial cells remains to be determined. Here we report the effect of specific blockade of the glial Krebs cycle and glutamine synthetase on the neuronal activity of the respiratory network. Application of 5 mM fluoroacetate, which selectively blocks the glial Krebs cycle, suppressed rhythmic respiratory burst activity. Substitution of either the Krebs cycle substrate isocitrate (3 mM) or glutamine (3 mM) restored rhythmic network activity. Blockade of glutamine synthetase by methionine sulfoximine (0.5 mM) suppressed rhythmic burst activity as well. Resubstitution of glutamine (3 mM) was able to restore rhythmic activity in the presence of methionine sulfoximine. This data demonstrates that the glutamate-glutamine cycle in astrocytes and their supply of glutamine to neuronal glutamatergic terminals is essential for the rhythm generation in the respiratory centre.