Background and Purpose Spreading depolarizations (SDs) may contribute to delayed cerebral ischemia after subarachnoid hemorrhage (SAH). We tested whether SD-inhibitor valproate reduces brain injury in an SAH rat model with and without experimental SD induction. Methods Rats were randomized in a 2x2 design and pretreated with valproate (200 mg/kg) or vehicle for 4 weeks. SAH was induced by endovascular puncture of the right internal carotid bifurcation. One day post-SAH, brain tissue damage was measured with T-2-weighted magnetic resonance imaging, followed by cortical application of 1 mol/L KCl (to induce SDs) or NaCl (no SDs). Magnetic resonance imaging was repeated on day 3 followed by histology to confirm neuronal death. Neurological function was measured with an inclined slope test. Results In the groups with KCl application, lesion growth between days 1 and 3 was 5773 mm3 in the valproate-treated versus 237 +/- 232 mm3 in the vehicle-treated group. In the groups without SD induction, lesion growth in the valproate- and vehicle-treated groups was 8 +/- 20 mm3 versus 27 +/- 52 mm3. On fitting a 2-way analysis of variance model, we found a significant interaction effect between treatment and KCl/NaCl application of 161 mm3 (P=0.04). Number and duration of SDs, mortality, and neurological function were not statistically significantly different between groups. Lesion growth on magnetic resonance imaging correlated to histological infarct volume (Spearman's rho =0.83; P=0.0004), with areas of lesion growth exhibiting reduced neuronal death compared with primary lesions. Conclusions In our rat SAH model, valproate treatment significantly reduced brain lesion growth after KCl application. Future studies are needed to confirm that this protective effect is based on SD inhibition.