Spontaneous cortical spreading depression (CSD) has been found to occur in the penumbral zone of the brain in rats with focal cerebral ischemia, and has been shown to promote expansion of infarction. Electrophysiological recording of CSD has been used for monitoring the penumbral zone, but with an inherently low spatial resolution; consequently, optical intrinsic signal imaging (OISI) was applied to characterize the spontaneous CSD waves following permanent left middle cerebral artery occlusion (MCAO) in rats under alpha-chloralose/urethane anesthesia. Besides the previous report about the regional variation of optical reflectance during spontaneous CSD following MCAO, the origin site of CSD was easily determined using OISI with the benefit of high resolution in the present study. Those origin points (n = 82) were dynamically located in the ipsilateral hemisphere cortex: sometimes outside of the 6 mm x 8 mm observation area in the parietal cortex (n = 19, 23%), and sometimes inside (n = 63, 77%). The data showed a general trend towards the medial cortex (0.40 +/- 0.15 mm per CSD). Because the lateral cortex of the rat brain proved to be infarcted with 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining after 4 h occlusion, the migration of the origin sites implied a growth of the infarcted area. Hence, the determination of origins of spontaneous CSD using OISI would contribute to the continued study of stroke.