An isolated brainstem-lung preparation of the newborn rat was developed for a pharmacological study on the respiratory reflexes in vitro. Spontaneous periodic depolarizations of the ventral roots (C4-C5) were observed at a frequency of 5-15/min and regarded as the respiratory activity. This activity was transiently inhibited when the lungs were inflated by gas pressure applied to the trachea. The inhibitory response of the respiratory rhythm was markedly depressed by the application of bicuculline or strychnine to the brainstem. These results suggest that gamma-aminobutyric acid and/or glycine may be neurotransmitters in the brainstem which cause the inhibitory respiratory reflex.