Patients with traumatic brain injury are typically maintained at low-normal levels of arterial partial pressure of carbon dioxide (PaCO2) to counteract the risk of elevated intracranial pressure during intensive care. However, several studies suggest that management at hypercarbic levels may have therapeutic benefit. Here we examined the impact of CO2 levels on spreading depolarizations (SD), a mechanism and marker of acute lesion development in stroke and brain trauma. In an acute preparation of mechanically ventilated (30/70 O2/N2) female rats, SDs were evoked by cortical KCl application and monitored by electrophysiology and laser doppler flowmetry; CO2 levels were adjusted by ventilator settings and supplemental CO2. During 90min of KCl application, rats were maintained at hypocapnia (end-tidal CO2 22±2mmHg) or hypercapnia (57±4mmHg) but did not differ significantly in arterial pH (7.31±0.10 vs. 7.22±0.08, p=0.31) or other variables. Surprisingly, there was no difference between groups in the number of SDs recorded (10.7±4.2 vs. 11.7±3.1; n=3 rats/group; p=0.75) nor in SD durations (64±27 vs. 69±37 sec, p=0.54). In separate experiments (n=3), hypoxia was induced by decreasing inhaled O2 to 10% and single SDs were induced under interleaved conditions of hypo-, normo-, and hypercapnia. No differences in SD duration were observed. In both normoxia and hypoxia experiments, however, mean arterial pressures were negatively correlated with SD durations (normoxia R2=-0.29; hypoxia R2=-0.61, p's<0.001). Our results suggest that any therapeutic benefit of elevated CO2 therapy may be dependent on an acidic shift in pH or may only be observed in conditions of focal brain injury.