The distribution of inspiratory and expiratory activities among rib-cage muscles was examined using isolated brainstem-spinal cord-rib preparations from neonatal rats. Expiratory activity was evoked by decreasing perfusate pH from 7.4 to 7.1. All internal intercostal muscles (IIMs) in the first to eleventh intercostal spaces showed expiratory bursts. Although the IIMs in the more caudal interspaces exhibited expiratory bursts for as long as the low pH solution was present in all preparations, the expiratory bursts obtained from the IIMs in the rostral interspaces gradually disappeared even under low pH conditions in about half the preparations, suggesting that the more caudal IIMs play the greater role in expiration. All thoracic ventral roots examined from T1VR-T11VR, but not T13VR, exhibited overt inspiratory bursts under normal pH conditions. Low pH solution induced additional expiratory bursts in all thoracic VRs. The ratio of the integral of the absolute electrical voltage during the expiratory phase to that during the inspiratory phase increased progressively and significantly from the rostral to the caudal interspaces. These results accord well with previous ones in mammals in vivo. Hence, the neuronal mechanisms necessary for a rostrocaudal gradient in spinal respiratory motor outputs seem to be preserved in this in vitro preparation.