The respiratory neural network in the mammalian medulla oblongata shows rhythmic activity before birth. GABA and glycine are considered to be involved in control of respiratory rhythm. Recently we have demonstrated respiratory failure in glutamic acid decarboxylase (GAD) 67-deficient mice [Tsunekawa N, Arata A, Obata K (2005) Development of spontaneous mouth/tongue movement and related neural activity, and their repression in mouse fetus lacking glutamate decarboxylase 67. Eur J Neurosci 21:173-178]. To further evaluate the involvement of GABA and glycine in fetal respiratory function, we studied neural activities in brainstem-spinal cord blocks prepared from GAD65-/-:67-/- and vesicular GABA transporter (VGAT)-/-mice on embryonic day 14 (E14)-E15 and E18. In these knockout mice, the synthesis of GABA and the vesicular release of GABA and glycine are completely absent, respectively. Spontaneous respiratory discharges were observed in the ventral roots at the cervical cord (C) 4 level from wild-type mice but not from the knockout mice on E18. Administration of substance P induced C4 discharges in GAD65-/-:67-/- preparations but not in VGAT-/- preparations. C4 discharges were observed in the knockout mice on E14-E15, although the frequency was lower than that in the wild-type. Neuronal activities in the respiratory network of the E18 brainstem were recorded using a "blind" patch-clamp technique. Expiratory and inspiratory neurons with their characteristic firing patterns were observed in the wild-type fetuses. Strychnine reversed inspiratory-phase hyperpolarization to large depolarization in expiratory neurons. On the other hand, neurons in the same area of the knockout mice fired spontaneously without any rhythm. Substance P induced hyperpolarizing potentials in medullary neurons of GAD65-/-:67-/- mice. Further administration of strychnine induced large depolarizing potentials. Rhythmic activities were not observed in VGAT-/- mice even in the presence of substance P and strychnine. These results indicate that the lack of GABA and glycine impairs the function of the respiratory network in mouse fetuses and the impairment progresses with fetal age.