This study investigated the effect of repetitive cortical spreading depression (CSD) on behaviour and the anatomical and physiological patterns of cellular activation of cortical and subcortical areas in awake, moving rats. Rat behaviours in response to repetitive CSD events evoked by the application of KCl were quantified with electrophysiological recording. Immunohistochemistry was used to quantify anatomical regions of cellular activation. The effects of acute valproic acid administration on the behavioural parameters and cellular activation were evaluated. CSD significantly decreased locomotor activity and induced freezing in awake, moving rats, and stimulated c-Fos expression in the cortex, trigeminal nucleus caudalis (TNC), and amygdala. CSD also resulted in a prominent increase in c-Fos expression in the ipsilateral thalamic reticular nucleus (TRN) visual sector. Electrophysiological recordings revealed propagation of CSD into the TRN. Valproic acid pretreatment decreased the duration of CSD-induced freezing episodes and reversed the CSD-induced reduction in locomotor activity. Acute valproic acid administration also significantly blocked CSD-induced c-Fos expression in the TNC and TRN. These findings show that CSD events cause consistent behavioural responses and activate specific brain regions in awake, freely moving rats. Selective activation of TRN by CSD and the suppression of this activation by valproic acid suggest that this brain region may play an important role in migraine pathogenesis and may represent a novel target for migraine therapy.