1. We examined whether expiratory activity can be observed when central chemoreceptors are activated by a decrement in the extracellular pH in an isolated brainstem-spinal cord-rib preparation from 0- to 3-day-old rats. Expiratory activity was defined as the burst activity that occurs in an internal intercostal muscle (IIM) during the silent period between the periodic inspiratory bursts in the C4 ventral root (which contains phrenic motor axons). 2. During perfusion with modified Krebs solution (26 mM HCO3-, 5 % CO2, pH 7.4), there was no consistent activity in IIM, though rhythmic inspiratory motor activity always appeared in the C4 ventral root. 3. When the pH of the perfusate was lowered from about 7.4 to 7.1 by reducing [HCO3-] from 26 to 10 mM, the frequency of the C4 inspiratory rhythm increased, and rhythmic activity appeared in IIM. In most cases, the rhythmic burst in IIM started just after the cessation of the C4 inspiratory burst and coincided with movement of the ribs in a caudal direction. This intercostal expiratory burst was limited to the first half of the expiratory phase. 4. The coordinated reciprocal motor activity between the C4 ventral root and IIM changed to a largely overlapping pattern when strychnine (5-10 microM), a glycine receptor antagonist, was added to the perfusate. 5. These results suggest (i) that the neuronal mechanisms responsible for expiratory motor activity are preserved in this in vitro preparation and (ii) that the glycinergic inhibitory system plays an important role in the coordination between inspiratory and expiratory motor activity during respiration.