Spreading depression (SD), a neuronal mechanism involved in brain pathophysiology, occurs in brain areas with high neuronal density such as the cerebral cortex. By contrast, the brainstem is thought to be resistant to SD. Here we show that DC shifts resembling cortical SD can be elicited in rat brainstem by topical application of KCl but not by pricking the brainstem. However, this was only possible until postnatal day 13, and, in addition, susceptibility for SD had to be enhanced. The latter was achieved by superfusion of the brainstem for 45 min with a solution containing acetate instead of chloride ions. Transient asphyxia or hypoxia by 2 min breathing 6% O₂ in N₂ had a similar effect. Negative brainstem DC deflections were paralleled by an increase of extracellular potassium concentration up to 40 mM and were spreading, but unlike cortical SD they were not inducible by glutamate and NMDA. Time course and slope of brainstem SD either resembled cortical SD, or were long-lasting and sustained. The latter stopped normal breathing. Different to cortical SD, negative brainstem DC deflections were changed in their slope (mostly converted into sustained shape, peak time was significantly prolonged, decline-time and duration were prolonged), but not abolished by the NMDA receptor blocker MK-801. Thus we demonstrate that the immature brainstem has the capacity to generate negative DC shifts which could be relevant as a risk factor in the newborn brainstem function.