In mammals, expiration is lengthened by mid-expiratory lung inflation (Breuer-Hering Expiratory reflex; BHE). The central pathway mediating the BHE is paucisynaptic, converging on neurones in the rostral ventrolateral medulla. An in vitro neonatal rat brainstem-lung preparation in which mid-expiratory inflation lengthens expiration was used to study afferent modulation of respiratory neurone activity. Recordings were made from respiratory neurones in or near the pre-Bötzinger Complex (preBötC). Respiratory neurone membrane properties and BHE-induced changes in activity were characterized. Our findings suggest the following mechanisms for the BHE: (i) lung afferent signals strongly excite biphasic neurones that convey these signals to respiratory neurones in ventrolateral medulla; (ii) expiratory lengthening is mediated by inhibition of rhythmogenic and (pre)motoneuronal networks; and (iii) pre-inspiratory (Pre-I) neurones, some of which project to abdominal expiratory motoneurones, are excited during the BHE. These findings are qualitatively similar to studies of the BHE in vivo. Where there are differences, they can largely be accounted for by developmental changes and experimental conditions.